Hemostatic agent for topical and internal use

ABSTRACT

This invention relates to deployable hemostatic materials comprising chitosan fibers. The hemostatic materials are suitable for use in sealing or controlling active bleeding from artery and vein lacerations and punctures, and for controlling oozing from tissue.

INCORPORATION BY REFERENCE TO RELATED APPLICATIONS

Any and all priority claims identified in the Application Data Sheet, orany correction thereto, are hereby incorporated by reference under 37CFR 1.57. This application is a continuation of U.S. application Ser.No. 11/061,243, filed on Feb. 18, 2005, which claims the benefit of U.S.Provisional Application No. 60/638,865 filed Dec. 22, 2004, U.S.Provisional Application No. 60/547,166 filed Feb. 23, 2004, and U.S.Provisional Application No. 60/547,257 filed Feb. 23, 2004. Each of theaforementioned applications is incorporated by reference herein in itsentirety, and each is hereby expressly made a part of thisspecification.

FIELD OF THE INVENTION

This invention relates to deployable hemostatic materials comprisingchitosan fibers. The hemostatic materials are suitable for use insealing or controlling active bleeding from artery and vein lacerationsand punctures, and for controlling oozing from tissue.

BACKGROUND OF THE INVENTION

Surgical procedures and traumatic injuries are often characterized bymassive blood loss. Conventional approaches such as manual pressure,cauterization, or sutures may be time consuming and are not alwayseffective in controlling bleeding.

Over the years, a number of topical hemostatic agents have beendeveloped to control bleeding during surgical procedures and to controlbleeding resulting from traumatic injury. Some agents such ascollagen-based powders, sponges, or cloths are of a particulate nature.Particulate hemostatic agents provide a lattice for natural thrombusformation, but are unable to enhance this process in coagulopathicpatients. Microfibrillar collagen, a particulate hemostatic agent, comesin powder form and stimulates the patient's intrinsic hemostaticcascade. However, this product has been reported to embolize and inducea localized inflammatory response if used during cardiopulmonary bypass.Pharmacologically-active agents such as thrombin can be used incombination with a particulate carrier, for example, as in a gelfoamsponge or powder soaked in thrombin. Thrombin has been used to controlbleeding on diffusely bleeding surfaces, but the lack of a frameworkonto which the clot can adhere has limited its use. The autologous andallogenic fibrin glues can cause clot formation, but do not adhere wellto wet tissue and have little impact on actively bleeding wounds.

SUMMARY OF THE INVENTION

A hemostatic material that is bioabsorbable, that provides superiorhemostasis, and that can be fabricated into a variety of forms suitablefor use in controlling bleeding from a variety of wounds is desirable.In addition the hemostatic material that is suitable for use in bothsurgical applications as well as in field treatment of traumaticinjuries is also desirable. For example, in vascular surgery, bleedingis particularly problematic. In cardiac surgery, the multiple vascularanastomoses and cannulation sites, complicated by coagulopathy inducedby extracorporeal bypass, can result in bleeding that can only becontrolled by topical hemostats. Rapid and effective hemostasis duringspinal surgery, where control of osseous, epidural, and/or subduralbleeding or bleeding from the spinal cord is not amenable to sutures orcautery, can minimize the potential for injury to nerve roots and reducethe procedure time. In liver surgery, for example, live donor livertransplant procedures or removal of cancerous tumors, there is asubstantial risk of massive bleeding. An effective hemostatic materialcan significantly enhance patient outcome in such procedures. Even inthose situations where bleeding is not massive, an effective hemostaticmaterial can be desirable, for example, in dental procedures such astooth extractions, as well as the treatment of abrasions, burns, and thelike. In neurosurgery, oozing wounds are common and are difficult totreat.

Accordingly, in a first aspect, a hemostatic material is provided, thematerial comprising chitosan fibers, wherein the chitosan has amolecular weight of about 1100 kDa or greater, and a degree ofacetylation of about 90% or greater.

In a preferred embodiment of the first aspect, the hemostatic materialcomprises a puff or a fleece.

In a preferred embodiment of the first aspect, the hemostatic materialcomprises a nonwoven fabric or a woven fabric.

In a preferred embodiment of the first aspect, the hemostatic materialcomprises a nonwoven fabric having a rough side and a smooth side.

In a preferred embodiment of the first aspect, the hemostatic materialcomprises a nonwoven fabric wherein both sides are rough.

In a preferred embodiment of the first aspect, the hemostatic materialcomprises a plurality of chitosan fiber layers.

In a preferred embodiment of the first aspect, the chitosan fibers havebeen treated with an acetic acid solution.

In a preferred embodiment of the first aspect, the chitosan fibers havebeen treated with glacial acetic acid.

In a second aspect, a process for preparing a hemostatic materialcomprising chitosan fibers is provided, wherein the chitosan has amolecular weight of about 1100 kDa or greater, and a degree ofacetylation of about 90% or greater, the process comprising providing afirst chitosan fiber layer; applying a weak acid to the first chitosanfiber layer; and placing a second chitosan fiber layer atop the firstchitosan fiber layer, whereby a hemostatic material is obtained.

In a preferred embodiment of the second aspect, the steps are repeatedat least once.

In a preferred embodiment of the second aspect, the process furthercomprises the step of heating the hemostatic material, whereby a liquidis vaporized from the hemostatic material.

In a preferred embodiment of the second aspect, the process furthercomprises compressing the hemostatic material between a first surfaceand a second surface; and heating the hemostatic material, whereby a dryhemostatic material is obtained.

In a preferred embodiment of the second aspect, the weak acid comprisesan acetic acid solution.

In a preferred embodiment of the second aspect, the weak acid comprisesglacial acetic acid.

In a preferred embodiment of the second aspect, the weak acid comprisesa solution of acetic acid having a pH of from about 3.0 to about 4.5.

In a third aspect, a method of treating a wound is provided, the methodcomprising the step of applying a hemostatic material to the wound,whereby bleeding or oozing is controlled, the hemostatic materialcomprising chitosan fibers, wherein the chitosan has a molecular weightof about 1100 kDa or greater, and a degree of acetylation of about 90%or greater.

In a preferred embodiment of the third aspect, the hemostatic materialis in a form selected from the group consisting of a puff, a sponge, anda fabric.

In a preferred embodiment of the third aspect, the wound is selectedfrom the group consisting of a tumor bed, a liver wound, and a brainwound.

In a preferred embodiment of the third aspect, the wound is selectedfrom the group consisting of an arterial puncture wound, a venouspuncture wound, arterial laceration wound, and a venous lacerationwound.

In a preferred embodiment of the third aspect, the chitosan fibers havebeen treated with glacial acetic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a scanning electron micrograph (SEM) image of a chitosanfleece.

FIG. 2 depicts sealing a femoral artery puncture with a hemostatic puff.The expandable, absorbable, biologically-compatible chitosan puff isplaced against the puncture wound via an incision in the skin. Thehemostatic sponge expands and holds itself in place against the wall ofthe artery, sealing the puncture.

FIG. 3 depicts a device for sealing an artery puncture with a hemostaticfleece.

FIG. 4 schematically depicts a process for obtaining chitosan fromshrimp waste.

FIG. 5 schematically depicts an apparatus for preparing chitosan fibers.

FIG. 6 provides a schematic of an assembly line for production ofchitosan fleece, including a feeder, loosen machine, carding machine,conveyer belt and winding machine.

FIG. 7 provides a scanning electron micrograph of chitosan fleece loadedwith 15% microporous polysaccharide microspheres.

FIG. 8 provides a scanning electron micrograph of chitosan fleece loadedwith 60% microporous polysaccharide microspheres.

FIGS. 9A and 9B provide scanning electron micrographs of microporouspolysaccharide microsphere clusters fused or bonded to chitosan fibers.

FIGS. 10A, 10B, and 10C provide SEM images of microporous polysaccharidemicrospheres physically loaded onto chitosan fleece.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The following description and examples illustrate a preferred embodimentof the present invention in detail. Those of skill in the art willrecognize that there are numerous variations and modifications of thisinvention that are encompassed by its scope. Accordingly, thedescription of a preferred embodiment should not be deemed to limit thescope of the present invention.

Hemostasis

Hemostasis is the arrest of bleeding, whether by normalvasoconstriction, by an abnormal obstruction, or by coagulation orsurgical means. Hemostasis by coagulation is dependent upon a complexinteraction of plasma coagulation and fibrinolytic proteins, platelets,and the blood vasculature. There are three categories of hemostasis:primary hemostasis, secondary hemostasis, and tertiary hemostasis.

Primary hemostasis is defined as the formation of the primary plateletplug. It involves platelets, the blood vessel wall and von Willebrandfactor. Injury to the blood vessel wall is initially followed byvasoconstriction. Vasoconstriction not only retards extravascular bloodloss, but also slows local blood flow, enhancing the adherence ofplatelets to exposed subendothelial surfaces and the activation of thecoagulation process. The formation of the primary platelet plug involvesplatelet adhesion followed by platelet activation then aggregation toform a platelet plug.

In platelet adhesion, platelets adhere to exposed subendothelium. Inareas of high shear rate, such as in the microvasculature, this ismediated by von Willebrand factor (vWf), which binds to glycoproteinIb-IX in the platelet membrane. In areas of low shear rate, such as inthe arteries, fibrinogen mediates the binding of platelets to thesubendothelium by attaching to a platelet receptor. The adhesion ofplatelets to the vessel wall activates them, causing the platelets tochange shape, to activate the collagen receptor on their surface, and torelease alpha and dense granule constituents. The activated plateletsalso synthesize and release thromboxane A2 and platelet activatingfactor, which are potent platelet aggregating agonists andvasoconstrictors.

Platelet aggregation involves the activation, recruitment, and bindingof additional platelets, which bind to the adhered platelets. Thisprocess in promoted by platelet agonists such as thromboxane 2, PAF,ADP, and serotonin. This activation is enhanced by the generation ofthrombin, another platelet agonist, through the coagulation cascade.Platelet aggregation is mediated primarily by fibrinogen, which binds toglycoprotein IIb/IIIa on adjacent platelets. This aggregation leads tothe formation of the primary platelet plug, is stabilized by theformation of fibrin.

In secondary hemostasis, fibrin is formed through the coagulationcascade, which involves circulating coagulation factors, calcium, andplatelets. The coagulation cascade involves three pathways: intrinsic,extrinsic, and common.

The extrinsic pathway involves the tissue factor and factor VII complex,which activates factor X. The intrinsic pathway involves high-molecularweight kininogen, prekallikrein, and factors XII, XI, IX and VIII.Factor VIII acts as a cofactor (with calcium and platelet phospholipid)for the factor IX-mediated activation of factor X. The extrinsic andintrinsic pathways converge at the activation of factor X. The commonpathway involves the factor X-mediated generation of thrombin fromprothrombin (facilitated by factor V, calcium and plateletphospholipid), with the production of fibrin from fibrinogen.

The main pathway for initiation of coagulation is the extrinsic pathway(factor VII and tissue factor), while the intrinsic pathway acts toamplify the coagulation cascade. The coagulation cascade is initiated bythe extrinsic pathway with the generation/exposure of tissue factor.Tissue factor is expressed by endothelial cells, subendothelial tissueand monocytes, with expression being upregulated by cytokines. Tissuefactor then binds to factor VII and this complex activates factor X.Factor X, in the presence of factor V, calcium and plateletphospholipid, then activates prothrombin to thrombin. This pathway israpidly inhibited by a lipoprotein-associated molecule, called tissuefactor pathway inhibitor. However, the small amount of thrombingenerated by this pathway activates factor XI of the intrinsic pathway,which amplifies the coagulation cascade.

The coagulation cascade is amplified by the small amounts of thrombingenerated by the extrinsic pathway. This thrombin activates theintrinsic pathway by activation of factors XI and VIII. Activated factorIX, together with activated factor VIII, calcium, and phospholipid,referred to as tenase complex, amplify the activation of factor X,generating large amounts of thrombin. Thrombin, in turn, cleavesfibrinogen to form soluble fibrin monomers, which then spontaneouslypolymerize to form the soluble fibrin polymer. Thrombin also activatesfactor XIII, which, together with calcium, serves to cross-link andstabilize the soluble fibrin polymer, forming cross-linked fibrin.

Tertiary hemostasis is defined as the formation of plasmin, which is themain enzyme responsible for fibrinolysis. At the same time as thecoagulation cascade is activated, tissue plasminogen activator isreleased from endothelial cells. Tissue plasminogen activator binds toplasminogen within the clot, converting it into plasmin. Plasmin lysesboth fibrinogen and fibrin in the clot, releasing fibrin and fibrinogendegradation products.

The preferred embodiments provide compositions and materials that reactwith the hemostatic system to treat or prevent bleeding. In particular,the compositions and materials of preferred embodiments result incoagulation of blood.

Effective delivery of hemostatic agents to wounds is particularlydesirable in the treatment of injuries characterized by arterial orvenous bleeding, as well as in surgical procedures where the control ofbleeding can become problematic, e.g., large surface areas, heavyarterial or venous bleeding, oozing wounds, and organlaceration/resectioning. The compositions and materials of preferredembodiments can possess a number of advantages in delivery of hemostaticagents to wounds, including but not limited to ease of application andremoval, bioadsorption potential, suturability, antigenicity, and tissuereactivity.

Depending upon the nature of the wound and the treatment methodemployed, the devices of preferred embodiments can employ differentforms. For example, a puff, fleece, or sponge form can be preferable forcontrolling the active bleeding from artery or vein, or for internalbleeding during laparoscopic procedures. In neurosurgery where oozingbrain wounds are commonly encountered, a sheet form of the hemostaticmaterial can be preferred. Likewise, in oncological surgery, especiallyof the liver, it can be preferred to employ a sheet form or sponge formof the hemostatic material, which is placed in or on the tumor bed tocontrol oozing. In dermatological applications, a sheet form can bepreferred. In closing punctures in a blood vessel, a puff form isgenerally preferred. A suture form, such as a microsuture or amacrosuture, can be preferred in certain applications. Despitedifferences in delivery and handling characteristic of the differentforms, the devices are effective in deploying hemostatic agents to anaffected site and to rapidly initiate hemostatic plug formation throughplatelet adhesion, platelet activation, and blood coagulation.

In preferred embodiments a hemostatic agent comprising chitosan fibersis employed. An auxiliary hemostatic agent, such as bioabsorbablemicroporous polysaccharide microspheres, can be deposited upon thechitosan fibers. However, any other suitable auxiliary hemostatic agentscan be employed.

Hemostatic Substrate

Any suitable hemostatic substrate can be employed as a support for thehemostatic agents of preferred embodiments. However, in a particularlypreferred embodiment, the hemostatic substrate comprises chitosan.Chitosan is obtained from chitin, a widely available biopolymer obtainedprincipally from shrimp and crab shell waste. Chitosan is the mainderivative of chitin, and is the collective term applied to deacetylatedchitins in various stages of deacetylation and depolymerization. Thechemical structure of chitin and chitosan is similar to that ofcellulose. The difference is that instead of the hydroxyl group as isbonded at C-2 in each D-glucose unit of cellulose, there is anacetylated amino group (—NHCOCH₃) at C-2 in each D-glucose unit inchitin and an amino group at C-2 in each D-glucose unit of chitosan.

Chitin and chitosan are both nontoxic, but chitosan is used more widelyin medical and pharmaceutical applications than chitin because of itsgood solubility in acid solution. Chitosan has good biocompatibility andis biodegradable by chitosanase, papain, cellulase, and acid protease.Chitosan exhibits anti-inflammatory and analgesic effects, and promoteshemostasis and wound healing. Chitosan has also been used as ahemostatic agent in surgical treatment and wound protection. Thehemostatic effect of chitosan has been described in U.S. Pat. No.4,394,373.

A single hemostatic substrate or combination of hemostatic substratescan be employed. Different substrate forms can be preferred, forexample, puff, fleece, fabric or sheet, sponge, suture, or powder. Ahomogeneous mixture of different substrate-forming materials can beemployed, or composite substrates can be prepared from two or moredifferent formed substrates. A preferred composite comprises chitosanand collagen.

A particularly preferred source of chitin for use in preparing chitosanfleece is crab shell. Chitin prepared from crab shell generally exhibitsa molecular weight that is much higher than the molecular weight ofchitin made from shrimp shell. Crab shell chitin also generally exhibitsa higher degree of deacetylation than shrimp shell chitin. Crab shellchitin typically exhibits an average molecular weight of from about600,000 to 1.3 million molecular weight. The degree of deacetylation isgenerally more than 90%, which can contribute to the higher molecularweight observed.

Preferred chitosan for use in preparing chitosan fiber has a molecularweight of greater than about 600, 650, 700, 750, 800, 850, 900, 950,1000, 1100, 1200, 1300, 1400, or 1500 kDa or more; more preferably fromabout 600, 650, 700, 750, 800, 925, 850, 875, 900, 925, 950, 975, 1000,1025, 1050, or 1075 kDa to about 1500 kDa; and most preferably fromabout 1100, 1125, 1150, 1175, 1200, 1225, 1250, or 1275 kDA to about1300, 1325, 1350, 1375, 1400, 1425, 1450, or 1475 kDa. The chitosanpreferably has a degree of acetylation of about 90, 91, 92, 93, 94, 95,96, 97, 98, or 99% or greater, more preferably from about 90.0, 90.5,91.0, 91.5, or 92.0% to about 92.5, 93.0, 93.5, 94.0, 94.5, or 95.0%.

While chitosan is generally preferred for use as a substrate, othersuitable substrates can also be employed. These substrates arepreferably bioabsorbable hydrophilic materials that can be fabricatedinto a desired form (fiber, sponge, matrix, powder, sheet, suture,and/or puff).

Other suitable substrates include a synthetic absorbable copolymer ofglycolide and lactide. This copolymer is marketed under the trade nameVICRYL™ (a Polyglactin 910 manufactured by Ethicon, a division ofJohnson & Johnson in Somerset, N.J.). It is absorbed though enzymaticdegradation by hydrolysis.

Gelatin sponge is an absorbable, hemostatic sponge used in surgicalprocedures with venous and oozing bleeding. The sponge adheres to thebleeding site and absorbs approximately forty five times its own weight.Due to the uniform porosity of the gelatin sponge, blood platelets arecaught within its pores, activating a coagulation cascade. Solublefibrinogen transforms into a net of insoluble fibrin, which stops thebleeding. When implanted into the tissue, the gelatin sponge is absorbedwithin three to five weeks.

Polyglycolic acid is a synthetic absorbable polymer also suitable foruse as a substrate. Polyglycolic acid is absorbed within a few monthspost-implantation due to its greater hydrolytic susceptibility.

Polylactide is prepared from the cyclic diester of lactic acid (lactide)by ring opening polymerization. Lactic acid exists as two opticalisomers or enantiomers. The L-enantiomer occurs in nature, a D,L racemicmixture results from the synthetic preparation of lactic acid. Fibersspun from polymer derived from the L-enantiomer have high crystallinitywhen drawn whereas fibers derived from the racemic mixture areamorphous. Crystalline poly-L-lactide is generally more resistant tohydrolytic degradation than the amorphous DL form, but can be increasedby plasticization with triethyl citrate, however the resulting productis less crystalline and more flexible. The time required forpoly-L-lactide to be absorbed by the body is relatively long compared toother bioabsorbable materials. High molecular weight poly-L-lactidepolymers can be prepared, and the fibers with large tensile strengthobtained.

Poly(lactide-co-glycolide) polymers are also suitable substrates. Thesecopolymers are amorphous between the compositional range 25 to 70 molepercent glycolide. Pure polyglycolide is about 50% crystalline, whereaspure poly-L-lactide is about 37% crystalline.

Polydioxanone can be fabricated into fibers to form a suitablesubstrate. Polycaprolactone, synthesized from e-caprolactone, is asemi-crystalline polymer absorbed very slowly in vivo. Copolymers ofe-caprolactone and L-lactide are elastomeric when prepared from 25%e-caprolactone, 75% L-lactide and rigid when prepared from 10%e-caprolactone, 90% L-lactide. Poly-b-hydroxybutyrate is a biodegradablepolymer that occurs in nature, that can easily be synthesized in vitro,and that is melt processable. Copolymers of hydroxybutyrate andhydroxyvalerate have more rapid degradation than can be achieved withpure poly-b-hydroxybutyrate.

Synthetic absorbable polyesters containing glycolate ester linkages aresuitable substrates. Similar copolymers prepared using dioxanone insteadof glycolide can also be employed, as can poly(amino acids).

Catgut, siliconized catgut, and chromic catgut can be suitable for useas substrates in certain embodiments. However, synthetic materials aregenerally preferred over natural materials due to their generallypredictable performance and reduced inflammatory reaction.

Hemostatic Agent

In certain embodiments, it can be desirable to add an auxiliaryhemostatic agent to the chitosan fiber hemostatic agents of preferredembodiments. Any suitable hemostatic agent can be deposited upon thesubstrates of preferred embodiments. However, in a particularlypreferred embodiment, the hemostatic agent comprises bioabsorbablemicroporous polysaccharide microspheres (for example, TRAUMADEX™marketed by Emergency Medical Products, Inc. of Waukesha, Wis.). Themicrospheres have micro-replicated porous channels. The pore size of thespheres enables water absorption and hyperconcentration of albumin,coagulation factors, and other protein and cellular components of theblood. The microspheres also impact platelet function and enhance fibrinformulation. In addition, the microspheres appear to accelerate thecoagulation enzymatic reaction rate. When applied directly, withpressure, to an actively bleeding wound, the particles act as molecularsieves to extract fluids from the blood. The controlled porosity of theparticle excludes platelets, red blood cells, and serum proteins largerthan 25,000 Daltons, which are then concentrated on the surface of theparticles. This molecular exclusion property creates a highconcentration of platelets, thrombin, fibrinogen, and other proteins onthe particle surface, producing a gelling action. The gelled, compactedcells and constituents accelerate the normal clotting cascade. Thefibrin network formed within this dense protein-cell matrix adherestightly to the surrounding tissue. The gelling process initiates withinseconds, and the resulting clot, while exceptionally tenacious, breaksdown normally along with the microparticles.

Other suitable hemostatic agents that can be employed in preferredembodiments include, but are not limited to, clotting factorconcentrates, recombinant Factor VIIa (NOVOSEVEN®); alphanate FVIIIconcentrate; bioclate FVIII concentrate; monoclate-P FVIII concentrate;haemate P FVIII; von Willebrand factor concentrate; helixate FVIIIconcentrate; hemophil-M FVIII concentrate; humate-P FVIII concentrate;hyate-C® Porcine FVIII concentrate; koate HP FVIII concentrate; kogenateFVIII concentrate; recombinate FVIII concentrate; mononine FIXconcentrate; and fibrogammin P FXIII concentrate. Such hemostatic agentscan be applied to the substrate in any suitable form (powder, liquid, inpure form, in a suitable excipient, on a suitable support, or the like).

A single hemostatic agent or combination of hemostatic agents can beemployed. Preferred loading levels for the hemostatic agent on thesubstrate can vary, depending upon the nature of the substrate andhemostatic agent, the form of the substrate, and the nature of the woundto be treated. However, in general it is desirable to maximize theamount of auxiliary hemostatic agent in relation to the substrate. Forexample, in the case of a hemostatic puff, a weight ratio of hemostaticagent to substrate of from about 0.001:1 or lower, 0.01:1, 0.05:1,0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, or 0.9:1 toabout 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1,1.9:1, 2:1 or higher is generally preferred, although higher or lowerratios can be preferred for certain embodiments.

Hemostatic Materials Comprising Chitosan Support

It is generally preferred to deposit an auxiliary hemostatic agent(e.g., microporous polysaccharide microspheres) onto a hemostaticsupport (e.g., chitosan fibers) to yield a hemostatic material to beapplied to a bleeding or oozing wound. However, in certain embodiments,chitosan fiber can be employed as an effective hemostatic materialwithout the addition of another hemostatic agent. For example, achitosan puff, as described below but without added microporouspolysaccharide microspheres, is a particularly effective hemostaticmaterial when applied to bleeding or oozing wounds. The various forms ofchitosan (fabric, sponge, suture, fiber, and the like) as describedherein, are also effective hemostatic materials in certain embodimentswhen employed without auxiliary agents. The preferred form of thehemostatic support can depend upon the application for which it is to beemployed.

Hemostatic Puff

Hemostatic puffs or fleece are a particularly preferred form, whereinthe substrate comprises a puff—a fibrous, cotton-like material that canbe manipulated into a suitable shape or size so as to accommodate aparticular wound configuration. FIG. 1 provides a scanning electronmicrograph image of a chitosan fleece. In a preferred embodiment, a puffis prepared from chitosan fibers as follows. Chitosan fibers preparedaccording to conventional methods were torn or cut (manually or bymechanical apparatus) into pieces and the pieces were flattened andlayered together. An acetic acid solution, glacial acetic acid, or otheracidic solution (preferably a solution of a pH from 3.0-4.5) is sprayedonto a first layer to secure the chitosan fibers to each other, therebyforming a net structure. Misting chitosan fleece or fibers with glacialacetic acid results in formation of the water soluble ammonium salt ofchitosan. The ammonium salt form of chitosan exhibits enhancedbioadhesion to wet tissues and increased hemostatic effect when comparedto untreated chitosan fleece or fibers. Acetic acid of a desiredconcentration misted onto chitosan fleece or fiber can act as a “glue”to adhere the fibers together, to adhere microporous polysaccharidemicrospheres to the fibers, or to better adhere the chitosan fleece towet tissues such as blood or body fluid.

Optionally, auxiliary agents in powder form, e.g., microporouspolysaccharide microspheres, can be sprayed onto the first chitosanfiber layer, and then another layer of chitosan fiber is placed on top.The deposition process (acidic solution followed by deposition ofanother chitosan fiber layer) is then repeated and the layers built upto a desired level. Agent in powder form can be added to the fiberlayers in a quantity sufficient to yield a puff comprising up to about50, 60, 70, 80, or 90% by weight or more of the auxiliary agent. Optimalloading level, if an auxiliary agent in powder form is added, can dependupon the application and the type of auxiliary agent employed. It isgenerally preferred not to add auxiliary agent or other powdersubstances to the top layer, but in certain embodiments it can bedesirable to do so. A preferred thickness for the fabric can be obtainedby selecting the total number of layers.

The resulting hemostatic material is dried in an oven under vacuum toyield a hemostatic puff. While it is generally preferred to employglacial acetic acid or an acetic acid aqueous solution, other acidicsolutions of similar pH or similar characteristics can also be employed.For example, lactic acid, citric acid, glycolic acid, and mixtures ofweak acids can be suitably employed. Likewise, any biologicallycompatible liquid that is a solvent for chitosan can also be employed,e.g., pure water, ethanolic solution, and the like. Such solutions canbe acidic, basic, or neutral. In certain embodiments, it can bepreferred to employ a solution that is not acidic. In such embodiments,another material in suitable form that provides adhesion betweenchitosan fibers can be employed, for example, gelatin, starch,carageenan, guar gum, collagen, pectin, and the like. While chitosan isa preferred substrate for preparing a hemostatic puff, other fibroussubstrates, particularly fibrous polysaccharide substrates, are alsosuitable for use.

By adjusting the moisture level in the chitosan fibers, adhesion betweenthe fibers and the loading capacity of any optional auxiliary agent canbe optimized. The liquid in the fibers assists in adhering the fibers toeach other and to compatible auxiliary agents. It can also be possibleto increase the loading capacity by employing thinner fibers. The fiberscan be of uniform thickness, or comprise a mixture of thicknesses.Thinner fibers can also adhere more firmly to an artery, vein, or otherwound.

In preparing a hemostatic puff comprising microporous polysaccharidemicrosphere loaded chitosan fibers, it is generally preferred that theresulting puff contain from about 0.1 to about 95 wt. % microporouspolysaccharide microspheres, more preferably from about 1 to about 60,65, 70, 75, 80, 85, 90, or 95 wt. % microporous polysaccharidemicrospheres, and most preferably from about 5, 10, 15, 20, or 25 wt. %to about 30, 35, 40, 45, 50, or 55 wt. microporous polysaccharidemicrospheres. In certain embodiments, however, higher or lower levels ofmicroporous polysaccharide microspheres can be preferred. If a differenthemostatic agent is employed, or other components are to be added to thechitosan fibers or other substrate, different loading levels can bepreferred.

To prepare a hemostatic chitosan puff that exhibits improvedexpandability, polyvinylalcohol (PVA) can be added to the acidicsolution. An acetic acid solution containing 2 wt. % PVA yields ahemostatic puff with enhanced elasticity, but also a puff that is lesshydrophilic. A high degree of hydrophilicity is generally preferred toensure that hemostatic puff adheres securely the wound. However, incertain embodiments the reduction in hydrophilicity can be relativelysmall and thus not significantly affect the ability of the puff toadhere to the wound.

Hemostatic Fabric

Hemostatic fabric can be prepared from chitosan fibers according to themethod described above for preparation of hemostatic puffs, with thefollowing modifications. One or more layers of chitosan fiber,optionally loaded with an auxiliary agent, are pressed flat and driedunder vacuum. It is generally preferred to use 2, 3, 4, 5, 6, 7, 8, 9,or 10 or more layers of chitosan fiber in preparing the fabric. It isgenerally preferred that one side of the fabric has a smooth surface andthe other side of the fabric have a rough surface (e.g., in the case ofchitosan, a TEFLON™ surface applied to a surface of the fiber layersduring heating yields a smooth side, while a release paper applied to asurface of the fiber layers yields a rough surface). However, in certainembodiments, a fabric having two rough sides can be preferred, such as,for example, for use in connection with an irregular wound, or a deepwound, such as a lethal groin injury. In preferred embodiments, therough surface is exposed to the wound so as to maximize contact of thechitosan fibers with the wound, resulting in an improved hemostaticeffect and superior adherence to the wound. In preparing a hemostaticfabric comprising chitosan fibers loaded with microporous polysaccharidemicrospheres, it is generally preferred that the resulting fabriccontain from about 0.01 to about 75 wt. % microporous polysaccharidemicrospheres, more preferably from about 1 to about 60 wt. % microporouspolysaccharide microspheres, and most preferably from about 5, 10, 15,20, or 25 wt. % to about 30, 35, 40, 45, 50, or 55 wt. % microporouspolysaccharide microspheres. In certain embodiments, however, higher orlower levels of microporous polysaccharide microspheres can bepreferred, or even no microspheres at all. If a different hemostaticagent is employed, or other components are to be added to the chitosanfibers or other substrate, different loading levels can be preferred.

The hemostatic fabric can be provided in the form of a sheet of apre-selected size. Alternatively, a larger sheet of hemostatic fabriccan be cut, trimmed, or folded to provide a size and shape appropriateto the wound. Although the hemostatic fabric is bioabsorbable, incutaneous or topical applications it can be removed from the wound aftera satisfactory degree of hemostasis is achieved, or it can be left inplace until the wound is healed. Hemostatic fabric can be useful asartificial skin, and/or can provide antibiotic properties. When thehemostatic fabric is employed in internal applications, it is preferablyleft in place to be absorbed by the body over time. Such hemostaticfabrics are particularly well suited for use in the treatment of oozingwounds, such as in tumor beds or brain tissue.

Hemostatic Sponge

A hemostatic sponge can be prepared according to methods known in theart for preparing a porous sponge from a biocompatible or bioabsorbablepolymeric material, e.g., chitosan. Such methods typically involvepreparation of a solution of the polymeric material, crosslinkingagents, and foaming agents. The sponge can be loaded with hemostaticagent at any convenient point or points in the process, e.g., duringformation of the sponge, or after preparation of the sponge.

Hemostatic Sutures

The hemostatic substrates of preferred embodiments can be fabricatedinto sutures. In a preferred embodiment, chitosan fibers are fabricatedinto microsutures. Processes for suture fabrication include extrusion,melt spinning, braiding, and many other such processes. The synthesis ofraw suture materials is accomplished by any number of processes knownwithin the textile industry. Suture sizes are given by a numberrepresenting diameter ranging in descending order from 10 to 1 and then1-0 to 12-0, with 10 being the largest and 12-0 being the smallest.Sutures can comprise monofilaments or many filaments twisted together,spun together, or braided. The sutures of preferred embodiments exhibitsatisfactory properties, including stress-strain relationship, tensilestrength, rate of retention, flexibility, intrinsic viscosity,wettability, surface morphology, degradation, thermal properties,contact angle of knots, and elasticity. Hemostatic sutures can beemployed in any suitable application. However, they are generally notpreferred for vessel anastamosis, since their hemostatic properties canresult in undesired clot formation within the vessel.

Hemostatic Powders

The hemostatic substrates of preferred embodiments can be formed into apowder and applied in such form directly to a wound. For example,chitosan particles, optionally combined with other materials, can beemployed as a void filler following tooth extraction.

Hemostatic Matrices

Three-dimensional porous matrices can be prepared from sintered polymerparticles, for example, chitosan particles, and medicaments ortherapeutic agents can be infused into the pores. Alternatively,microcapsules comprising a chitosan shell encapsulating a medicament ortherapeutic agent can be sintered to form a matrix.

Wound Dressings

While it is generally preferred to apply the hemostatic material (forexample, a hemostatic fabric, sponge, puff, or powder prepared asdescribed above) directly to the wound, and while the hemostaticmaterial exhibits satisfactory adhesion to many types of wounds, incertain embodiments it can be preferred to incorporate the hemostaticmaterial into a wound dressing including other components.

To ensure that the hemostatic material remains affixed to the wound, asuitable adhesive can be employed, for example, along the edges of oneside of the hemostatic fabric, sponge or puff. Although any adhesivesuitable for forming a bond with skin can be used, it is generallypreferred to use a pressure sensitive adhesive. Pressure sensitiveadhesives are generally defined as adhesives that adhere to a substratewhen a light pressure is applied but leave no residue when removed.Pressure sensitive adhesives include, but are not limited to, solvent insolution adhesives, hot melt adhesives, aqueous emulsion adhesives,calenderable adhesive, and radiation curable adhesives. Solutionadhesives are preferred for most uses because of their ease ofapplication and versatility. Hot melt adhesives are typically based onresin-tackified block copolymers. Aqueous emulsion adhesives includethose prepared using acrylic copolymers, butadiene styrene copolymers,and natural rubber latex. Radiation curable adhesives typically consistof acrylic oligomers and monomers, which cure to form a pressuresensitive adhesive upon exposure to ultraviolet lights.

The most commonly used elastomers in pressure sensitive adhesivesinclude natural rubbers, styrene-butadiene latexes, polyisobutylene,butyl rubbers, acrylics, and silicones. In preferred embodiments,acrylic polymer or silicone based pressure sensitive adhesives are used.Acrylic polymers generally have a low level of allergenicity, arecleanly removable from skin, possess a low odor, and exhibit low ratesof mechanical and chemical irritation. Medical grade silicone pressuresensitive adhesives are preferred for their biocompatibility.

Amongst the factors that influence the suitability for a pressuresensitive adhesive for use in wound dressings of preferred embodimentsare the absence of skin irritating components, sufficient cohesivestrength such that the adhesive can be cleanly removed from the skin,ability to accommodate skin movement without excessive mechanical skinirritation, and good resistance to body fluids.

In preferred embodiments, the pressure sensitive adhesive comprises abutyl acrylate. While butyl acrylate pressure sensitive adhesives aregenerally preferred for many applications, any pressure sensitiveadhesive suitable for bonding skin can be used. Such pressure sensitiveadhesives are well known in the art.

As discussed above, the hemostatic materials of preferred embodimentsgenerally exhibit good adherence to wounds such that an adhesive, forexample, a pressure sensitive adhesive, is not necessary. However, forease of use and to ensure that the hemostatic material remains in afixed position after application to the wound, it can be preferable toemploy a pressure sensitive adhesive.

While the hemostatic fabrics and other hemostatic materials of preferredembodiments generally exhibit good mechanical strength and woundprotection, in certain embodiments it can be preferred to employ abacking or other material on one side of the hemostatic material. Forexample, a composite including two or more layers can be prepared,wherein one of the layers is the hemostatic material and another layeris, e.g., an elastomeric layer, gauze, vapor-permeable film, waterprooffilm, a woven or nonwoven fabric, a mesh, or the like. The layers canthen be bonded using any suitable method, e.g., adhesives such aspressure sensitive adhesives, hot melt adhesives, curable adhesives,application of heat or pressure such as in lamination, physicalattachment through the use of stitching, studs, other fasteners, or thelike.

Interaction between Chitosan Fibers and Wound

Chitosan fibers of preferred embodiments exhibits a hemostatic effectwhen placed in a bleeding or oozing wound. The physical and chemicalcharacteristics chitosan fiber were examined to determine the mechanismof their hemostatic action and to maximize their hemostatic effect.While not wishing to be bound to any particular theory, and while themechanism of chitosan's hemostatic function has not yet beendefinitively elucidated, it is believed that its calcium content plays asignificant role in hemostasis. The calcium can stimulate platelets torelease b-TG, PF-4 factors, or other substances involved in thehemostasis process.

The content of calcium in a 0.2 g sample of chitosan (prepared bydeacetylation, 91.8 wt. % purity, 1000 kD molecular weight) was measuredat 0.238 wt. %. Calcium content was measured by Inductively CoupledPlasma (ICP) quantometer measurements performed with a Jarrell-Ash-1100ICP-Auger Electron Spectroscopy (AES) instrument at a pressure of1.6-105 Pa, under an argon atmosphere. Sample preparation involved, as afirst step, dissolving the chitosan fiber in concentrated nitric acid.The solution was allowed to stand for two hours, after which it wasboiled until all of the water in solution was vaporized. The residueremaining was dissolved in a 100 ml volume of 1 M nitric acid, andcalcium content was then determined.

The literature suggests that the hemostatic effect of chitosan may notfollow the coagulation cascade pathways as described above, becausechitosan can still cause coagulation of blood from which all of theplatelets, white blood cells, and plasma have been removed. Chitosan'shemostatic effect is most likely due to its ability to causeerythrocytes to coalesce to each other, thereby forming a blood clot.When chitosan fibers come into contact with blood, the blood penetratesinto the network formed by chitosan fibers. Chitosan is hydrophilic andis wettable to form a hydrogel. The porous hydrogel can either absorbblood cells or provide enough space for the blood cells to diffuse intoit. These factors can induce hemostasis by causing erythrocytes tocoalesce and to form a blood clot. Another hypothesis is that chitosan,a naturally positively charged polysaccharide, can interact withnegative charges on the surface of blood proteins to cause erythrocytesto coalesce to each other.

Chitosan is hydrophilic and biodegradable, and exhibits biocompatibilityhemostatic properties. It is easily and effectively combined with othermaterials, such as microporous polysaccharide microspheres, and exhibitsstrong physical adsorption and adhesion amongst fibers. Chitosan alsobonds strongly to microporous polysaccharide microspheres, possibly dueto the similarity in their skeletal chemical structures, both of whichare based on glucose units. Chitosan has a strong affinity to cells,thereby resulting in an effective hemostatic material.

The loading efficiency of microporous polysaccharide microspheres in apuff comprising chitosan fibers was determined. Loading efficiencies ofup to 90% can be achieved while maintaining the pliability of the puff.At loading efficiencies above 90%, hardening of the puff can result, butcan be acceptable in certain embodiments.

When chitosan fibers are loaded or combined with another polysaccharidematerial, such as microporous polysaccharide microspheres, variousadhering or bonding mechanisms can be involved. In one mechanism,electrostatic forces maintain contact between the fibers, between themicroporous polysaccharide microspheres, or between the microporouspolysaccharide microspheres and the chitosan fibers. In anothermechanism, the particles are held in place by physical forces, with thechitosan fiber forms a lattice or matrix that supports the microporouspolysaccharide microspheres. In yet another mechanism, an acidicsolution added to the chitosan fibers and the microporous polysaccharidemicrospheres causes components to solubilize and bond together.

While chitosan fibers can be bonded or adhered each other (or tomicroporous polysaccharide microspheres) by any of the three methodsreferred to above (electrostatic forces, physically, or chemically), itis generally preferred to employ a combination of two or more differentmechanisms, so as to produce optimal loading of the fleece, for example,static and physical, static and chemical, physical and chemical, orstatic and physical and chemical.

The expansion of microporous polysaccharide microspheres and chitosanafter they contact water was measured. It was observed that puremicroporous polysaccharide microspheres absorb water and expand togenerate pressure against surrounding structures. However, there was noclinically significant expansion of microporous polysaccharidemicrospheres deposited on a chitosan fiber puff upon contact with water.The measurements were conducted as follows: 19 g of TRAUMADEX™microporous polysaccharide microspheres were placed in a device, thediameter of which was 1.55 cm, to measure expansion. Water was added tothe TRAUMADEX™, resulting in the water's adsorption. Weight was added tothe top of the device to prevent TRAUMADEX™ from expanding. The weightadded corresponds to the pressure that TRAUMADEX™ produces after itcontacts water. In the experiment, the difference in the weight appliedbefore contact of the TRAUMADEX™ with water and the weight applied aftercontact of the TRAUMADEX™ with water was 270 g. Accordingly, thepressure which TRAUMADEX™ exerted after it contacted water was 107 mmHg. The same method was employed to measure the expansion of TRAUMADEX™deposited on a chitosan puff, but the volume change observed was toosmall to be measured. It is believed that the porous chitosan puffprovides sufficient space for the expanded TRAUMADEX™ such that nosignificant volume change of the TRAUMADEX™ deposited on the chitosanpuff can be detected upon contact with water.

Closure of Femoral Artery Puncture Wounds

A hemostatic puff was developed for use in conjunction with a femoralartery puncture wound closure device, described in copending U.S. patentapplication Ser. No. 10/463,754, filed Jun. 16, 2003, and entitled“VASCULAR WOUND CLOSURE DEVICE AND METHOD”, the contents of which ishereby incorporated by reference in its entirety. FIG. 2 depicts sealinga femoral artery puncture with a hemostatic puff. The hemostatic puffcan be wrapped around the blood indication catheter of the wound closuredevice, depicted in FIG. 3, so as to be efficiently and effectivelydelivered to the top of the puncture wound. The vascular wound closuredevice 300 depicted in FIG. 3 comprises a catheter 310 having a proximalend and a distal end defining a lumen therebetween, and can be used forplacing a hemostatic puff 270 to the top of a puncture wound. A couplingmember 320 is movably disposed about the catheter 310 and is configuredto mechanically couple to the stop member 316. The device has a handle340 and handle 362 and a delivery tube 350. In a particularly preferredembodiment, the hemostatic puff, optionally with an adhesive or othersubstance providing enhanced adhesion to the wound, are delivered to thewound by the wound closure device.

In a venous laceration, the conventional method of repairing thelaceration involves temporarily stopping the bleeding, occluding thevein, suctioning out the blood, then suturing or clipping the lacerationto repair it. A vessel patch can also be required in conventionalmethods. The hemostatic fabrics of preferred embodiments can also beemployed to treat venous or arterial lacerations merely by compressingthe fabric to the laceration and allowing it to remain in place andeventually be absorbed by the body.

Closure of Wounds Using Endoscopic Devices

The chitosan fleece of preferred embodiments is particularly suitablefor use in connection with endoscopic or luminal devices, especiallyendoscopic devices for use in gastrointestinal applications, forcontrolling bleeding. Chitosan fleece can be manipulated into a smallpuff, which can be inserted through the lumen of the endoscope to beplaced on a wound, or to treat bleeding. For example, a 2 mm diameterpuff of chitosan fleece, or a puff of another size suitable to the innerdiameter of the lumen of the device, can be inserted through theendoscope and used to control gastrointestinal or other bleeding, e.g.,bleeding from ulcers, tumors, or lesions, or bleeding resulting fromsurgical procedures such as tumor removal.

Preparation of Chitosan

Chitin is present in crustacean shells as a composite with proteins andcalcium salts. Chitin is produced by removing calcium carbonate andprotein from these shells, and chitosan is produced by deacetylation ofchitin in a strong alkali solution. U.S. Pat. No. 3,533,940, thecontents of which are incorporated by reference herein in its entirety,describes a method for the preparation of chitosan.

A preferred method for obtaining chitosan from crab or other crustaceanshells is as follows. Calcium carbonate is removed by immersing theshell in dilute hydrochloric acid at room temperature for 24 hours(demineralization). Proteins are then extracted from the decalcifiedshells by boiling them with dilute aqueous sodium hydroxide for sixhours (deproteinization). The demineralization and deproteinizationsteps are preferably repeated at least two times to remove substantiallyall of the inorganic materials and proteins from the crustacean shells.The crude chitin thus obtained is washed then dried. The chitin isheated at 140° C. in a strong alkali solution (50 wt. %) for 3 hours.Highly deacetylated chitosan exhibiting no significant degradation ofmolecular chain is then obtained by intermittently washing theintermediate product in water two or more times during the alkalitreatment.

A process for obtaining chitosan from shrimp waste is schematicallydepicted in FIG. 4.

Preparation of Chitosan Fiber

In a preferred embodiment, a wet spinning method is employed to preparechitosan fiber. First, chitosan is dissolved in a suitable solvent toyield a primary spinning solution. Preferred solvents include acidicsolutions, for example, solutions containing trichloroacetic aceticacid, acetic acid, lactic acid, and the like, however any suitablesolvent can be employed. The primary spinning solution is filtered anddeaerated, after which it is sprayed under pressure into a solidifyingbath through the pores of a spinning jet. Solid chitosan fibers arerecovered from the solidified bath. The fibers can be subjected tofurther processing steps, including but not limited to drawing, washing,drying, post treatment, functionalization, and the like. FIG. 5schematically depicts an apparatus for preparing chitosan fibers. Theapparatus includes a dissolving kettle 1, a filter 2, a middle tank 3, astorage tank 4, a dosage pump 5, a filter 6, a spinning jet 7, asolidifying bath 8, a pickup roll 9, a draw bath 10, a draw roll 11, awashing bath 12, and a coiling roll 13.

A preferred method for preparing chitosan fiber suitable for fabricationinto the hemostatic materials of preferred embodiments is as follows.The primary chitosan spinning solution is prepared by dissolving 3 partschitosan powder in a mixed solvent at 5° C. containing 50 partstrichloroacetic acid (TDA) to 50 parts methylene dichloride. Theresulting primary spinning solution is filtered and then deaerated undervacuum. A first solidifying bath comprising acetone at 14° C. isemployed. The aperture of the spinning jet is 0.08 mm, the hole count isforty-eight, and the spinning velocity is 10 m/min. The spinningsolution is maintained at 20° C. by heating with recycled hot water. Thechitosan fibers from the acetone bath are recovered and conveyed via aconveyor belt to a second solidifying bath comprising methanol at 15° C.The fibers are maintained in the second solidifying bath for tenminutes. The fibers are recovered and then coiled at a velocity of 9m/min. The coiled fibers are neutralized in a 0.3 g/l KOH solution forone hour, and are then washed with deionized water. The resultingchitosan fiber is then dried, after which it is ready for fabricationinto the hemostatic materials of preferred embodiments.

In a particularly preferred embodiment, glacial, or anhydrous, aceticacid is employed as an agent to adhere the chitosan fibers to each otherin embodiments where chitosan fibers, either alone or with an addedmedicament, therapeutic agent or other agent, are used in forming ahemostatic agent. In addition to providing good adherence between thechitosan fibers, fibers treated with glacial acetic acid also exhibitexceptional ability to adhere to wounds, including arterial or femoralwounds.

Depending upon the application, the concentration of acetic acid insolution can be adjusted to provide the desired degree of adhesion. Forexample, it can be desirable to employ a reduced concentration of aceticacid if the chitosan fibers are to be employed in treating a seepingwound or other wound where strong adhesion is not desired, or inapplications where the hemostatic agent is to be removed from the wound.In such embodiments, an acetic concentration of from about 1 vol. % orless to about 20 vol. % is generally employed, and more preferably aconcentration of from about 2, 3, 4, 5, 6, 7, 8, 9, or 10 vol. % toabout 11, 12, 13, 14, 15, 16, 17, 18, or 19 vol. % is employed. Wherestrong adhesion between fibers, or strong adhesion to the wound isdesired, a concentration greater than or equal to about 20 vol. % ispreferred, more a preferably from about 50, 55, 60, 65, or 70 vol. % toabout 75, 80, 85, 90, 95, or 100 vol. %, and most preferably from about95, 96, 97, 98, or 99 vol. % to about 100 vol. %.

Experiments Preparation of Chitosan Fleece

Chitosan fleece can be prepared using equipment commonly employed in thetextile industry for fiber production. A typical assembly line forproduction of chitosan fleece can employ a feeder, a loosen machine, acarding machine, a conveyor belt, and lastly a winding machine, asdepicted in FIG. 6. In the feeder, chitosan short fiber is fed through afeeder and into a loosen machine, wherein chitosan short fiber isloosened by several beaters. In the carding machine, chitosan fibers areripped and turned into chitosan fleece by high speed spinning of acylinder and roller pin, then the fleece is peeled off as a separatedthin layer of net by a duffer. A thin layer of chitosan fleece netseparated from the duffer moves on the conveyer belt. A controlledaqueous solution of acetic acid is sprayed on the chitosan fleece andthen a specified amount of microporous polysaccharide microspheres isdistributed homogeneously while the chitosan fleece net is moving on theconveyer belt. The chitosan fleece loaded with microporouspolysaccharide microspheres is then collected by a reel and then is sentto a vacuum oven for drying.

Characterization of Chitosan Fiber

Determination of the Water Content 1.0 g chitosan fabric puff (W₁) wasaccurately weighted in a clean and dried beaker, and then the beaker wasremoved into an oven at 100° C. for 12 hours. The dried chitosan samplewas weighted again (W₂), and then the water content was calculated asfollowing formula:

α=(W ₁ −W ₂)/W ₂

Determination of Average Molecular Weight

Dried chitosan of 0.3 g was accurately weighted and dissolved in 0.1 molL⁻¹ CH₃COONa-0.2 mol L⁻¹ CH₃COOH solution. Five different concentrationsof chitosan solutions were prepared. The relative viscosity was measuredat 25±0.5° C. in a constant temperature water bath with a Ubbelohdeviscometer. Intrinsic viscosity is defined as:

[η]C(η_(red))→0

and is obtained by extrapolating the reduced viscosity versusconcentration data to zero concentration. See, e.g., Qurashi T, Blair HS, Allen S J, J. Appl. Polym. Sci., 46:255 (1992). The intercept on theordinate is the intrinsic viscosity. The viscosity average-molecularweight was calculated based on the Mark-Houwink equation as follows:

[η]=KM ^(α)

where K==1.81×10⁻³ L/g, α=0.93.

Determination of the Degree of Deacetylation

The measurements were made by the modified method reported byBroussignac, Chem. Ind. Genie. Chim. 99:1241 (1969). Dried chitosan inan amount of 0.3 g was accurately weighted and dissolved in 0.1 mol L⁻¹HCl. The solution was titrated with 0.1 mol L⁻¹ NaOH using bromocresolgreen as an indicator. The degree of deacetylation was calculated asfollow:

${N\; H_{2}\mspace{14mu} \%} = {\frac{( {{C_{1}V_{1}} - {C_{2}V_{2}}} ) \times 0.016}{G( {100 - W} )} \times 100}$

wherein C₁ is the concentration of HCl (mol L⁻¹); C₂ is theconcentration of NaOH (mol L⁻¹); V₁ is the volume of HCl (ml); V₂ is thevolume of NaOH (ml); G is the sample weight; W is the water percentageof sample (%); and 0.016 is the weight of NH₂ equal to 1 ml 0.1 mol L⁻¹HCl (g).

Degree of deacetylation (%)=NH₂%/9.94%×100%

wherein 9.94% is the theoretical NH₂ percentage when 100% of theCH₃CONH— group was deacetylated.

Determination of Heavy Metal Contents (Cr, Cu, Zn, Pb, Hg)

The Cr, Cu, Zn, and Pb contents were determined by an InductivelyCoupled Plasma Atomic Emission Spectrometry (ICP-AES) method. Beforemeasurement, the chitosan sample was treated as follows: a 0.1 gchitosan sample was accurately weighed, and then was soaked into acrucible which contained 2 mL of 90% nitric acid and 0.6 ml of 50%HClO₄. The chitosan sample was gradually dissolved in the mixed acidsolution, and simultaneously underwent oxidative degradation. Afterreacting for 2 hrs, the dissolved sample was slowly heated to slowlyvaporize all of the liquid, and then was combusted by a burner until allthe organic components decomposed and disappeared. The residuecontaining inorganic salts was diluted by a 2% nitric acid solution to10 mL and then the heavy metal contents were measured by ICP-AES(Jarrell-Ash 1100+2000 ICP-AES).

The mercury content in chitosan was determined by Atomic AbsorptionSpectrometry (AAS). A sample of 0.1 g accurately weighed chitosan wasdissolved in 10 mL 2% nitric acid. Mercury content of this sample wasthen measured by AAS.

Analysis of Chitosan Sample 1 yielded the following results: watercontent: 11.75%; molecular weight: 1326 kDa; degree of deacetylation:91.2%; metal content (μg/g): Al 59.1, Ba 2.95, Ca 187, Cr 8.86, Cu 3.76,Fe 34.9, Mg 59.1, Na 169, Si 185, Pb 16.3, Zn 25.5, Hg 0.16.

Analysis of Chitosan Sample 2 yielded the following results: watercontent: 12.38%; molecular weight: 1407 kDa; degree of deacetylation:93.6%; metal content (μg/g): Al 106, Ba 4.36, Ca 751, Cr 12.6, Cu 40.7,Fe 116, Mg 319, Na 193, Si 973, Pb 8.76, Zn 8.47×10³, Hg 0.16

The quality of the chitosan fiber tested was acceptable, based on theresults of analysis of Chitosan Samples 1 and 2, each of which had anaverage molecular weight >1000 kDa (1 million), a degree ofdeacetylation >90%, and a harmful heavy metal (Cr, Cu, Pb, Hg) contentthat was very low (except for Zn content in Chitosan Sample 2).

Loading Percentage of Microporous Polysaccharide Microspheres and theQuality of the Chitosan Fleece

Increased loading percentage of microporous polysaccharide microspherescan be achieved by utilizing two-step manufacturing processes. The firststep is to loosen the chitosan fibers and the second step is to card theloosened chitosan fibers into a thin layer of chitosan fleece. Thequality of chitosan fleece can directly affect the loading percentage ofa hemostatic agent such as microporous polysaccharide microspheres,which in turn can affect the fleece's hemostatic function. Themicroporous polysaccharide microspheres loading percentage of samplescan be increased from less than or equal to 25 wt. % up to 30-40% whenthe above-described two-step process is employed.

Preparation of Chitosan Puff

A hemostatic puff can be prepared from chitosan fibers by building uplayers of chitosan fiber and microporous polysaccharide microspheres“glued” together using an acetic acid solution, which are then driedunder vacuum. A “glue” solution was prepared comprising an acetic acidsolution with a pH value of from 3.0 to 4.5. The chitosan fibers weretorn into pieces. After laying down a first layer of chitosan pieces,the acetic acid solution was sprayed onto the chitosan pieces, and thenthe microporous polysaccharide microspheres were added. A second layerwas formed upon the first layer by the same procedure. Layers were builtup in this fashion until seven layers were obtained, except that nomicroporous polysaccharide microspheres were added to the topmost layer.The acetic acid solution acted not only as a glue between chitosanlayers, but also increased the hemostatic powder's ability to adhere tothe chitosan fibers. Alternatively, a hemostatic puff comprising onlychitosan fibers can be prepared according to the method described aboveby omitting the steps of adding microporous polysaccharide microspheresto the chitosan layers. Loading efficiency for microporouspolysaccharide microspheres in hemostatic puffs prepared as describedabove is provided in Table 1.

TABLE 1 Drug Loading Efficiency of Chitosan (CS) Puff CS weight (g)after Drug CS + drug Loading Fiber drying/before drying (g) (afterdrying) (g) efficiency Condition 1.96/(2.19) 0 1.96 — loose/flexible1.92/(2.15) 0.25 2.15 92.0% loose/flexible 1.82/(2.03) 0.51 2.28 90.1%loose/flexible 1.98/(2.21)* 1.01 2.96 97.0% hard *Two times as muchwater was sprayed onto the fibers compared to that used in the otherexamples.

This hemostatic chitosan puffs thus prepared exhibited good hemostaticfunction and swelling ability. When placed on or in a wound, the puffsabsorbed the blood immediately. The blood would pass through the firstfew chitosan layers, then immediately solidify to prevent furtherbleeding. Such hemostatic chitosan puffs biodegrade to nontoxicmaterials in the body after a period time, thus surgery is not needed toremove the puff if it is placed internally.

To improve the elasticity of the hemostatic puff, the chitosan fiberswere modified with CELVOL™ 205 PVA (manufactured by Celanese Ltd. ofDallas, Tex., partially hydrolyzed polymer of acetic acid ethenyl esterwith ethenol) to decrease their hydrophilicity. The procedure forpreparing PVA modified hemostatic puff is similar to the procedure forpreparing unmodified puff, the primary difference being that PVA isadded to acetic acid solution applied to the chitosan layers. Theconcentration of PVA in the acetic solution was 2 wt. %. The PVAmodified puff exhibited slightly improved elasticity when compared tothe unmodified puff.

Preparation of Chitosan Sponge

A hemostatic sponge is prepared according to the following procedure.Chitosan and PVA are dissolved in dilute acetic acid solution. Palladiumchloride is added as a catalyst and toluene diisocyanate is added as afoaming agent and cross-linking agent. The crude product is washed withammonia solution and dried in an oven. The sponge is optionally loadedwith a medicament or therapeutic agent either by adding the hemostaticagent to the acetic acid solution before preparation of the sponge, orby loading it into the sponge after it is prepared.

Preparation of Chitosan Fabric

Hemostatic fabric was prepared according to the following procedure.First, an aqueous solution of 1 wt. % acetic acid with a pH of 3.0 wasprepared. Chitosan fiber was separated into pieces and homogeneouslylaid on a glass plate covered with releasing paper to form a thin layer.The aqueous acetic acid solution was sprayed onto the chitosan fibersurface, and a specified amount of hemostatic powder was distributedover the chitosan fiber. Additional layers were built up by the sameprocedure. After a small amount of aqueous acetic acid solution wassprayed onto the uppermost chitosan fiber layer, a flat plate ofpolytetrafluoroethylene (TEFLON™) was placed on the uppermost chitosanfiber layer. Samples comprising five layers were thus prepared.

The entire system was placed in a vacuum oven and dried under vacuum forthree hours at 50° C. The TEFLON™ plate and releasing paper wereremoved, and the non-woven hemostatic fabric was recovered. The upperlayer which was in contact with the TEFLON™ plate was covered with athin membrane of chitosan, and the bottom layer which was in contactwith the releasing paper was made up of nonwoven fibrous chitosan havinga rough surface.

Microscopy of Chitosan Fleece Loaded with Microporous PolysaccharideMicrospheres

Chitosan fleece loaded with different amounts of microporouspolysaccharide microspheres were examined using scanning electronmicroscopy. At 15% loading, uneven distribution of the microporouspolysaccharide microspheres on chitosan fleece was observed (FIG. 7). At60% loading, fewer microporous polysaccharide microspheres are observed(FIG. 8). It is believed that microporous polysaccharide microsphereswere dislodged from the loaded chitosan fleece before or during theprocess of obtaining the SEM image. In the 60% loaded specimen,particulate microporous polysaccharide microspheres on the surface ofthe specimen container were observed. It is also possible that themicroporous polysaccharide microspheres were removed by the vacuum usedin the SEM sputter coating process to prepare the specimens. It isbelieved that when the loose microporous polysaccharide microspheres aredeposited in the interstices of the chitosan fleece, some weakelectrostatic bonding of the neutral or slightly acidic microporouspolysaccharide microspheres to the cationic chitosan occurs. FIGS. 10A,10B, and 10C provide SEM images of microporous polysaccharidemicrospheres physically loaded onto chitosan fleece. The loaded fleecewas pressed onto a carbon sticky tape. As seen in the images, many ofthe spheres were dislodged during the process of transferring the loadedfleece onto the sticky tape, but retain their pore morphology.

The attachment of the microporous polysaccharide microspheres to thestrands of chitosan fleece was also observed. Some degree of melting orfusing of both the chitosan and microporous polysaccharide microspherescan also occur during the manufacturing process, resulting in a bondthat can be visualized with scanning electron microscopy. It wasobserved that the microporous polysaccharide microspheres attach to thestrands in clusters, with one or more microspheres fusing to the strand.These fused spheres generally exhibit a smooth surface and do not havethe detailed pore structure of spheres in the clusters not directlyattached to the strand. FIGS. 9A and 9B provide scanning electronmicrographs of microporous polysaccharide microsphere clusters fused orbonded to chitosan fibers with loss of pore structure. Once themicroporous polysaccharide microspheres lose their pores, as can occurduring fusing or melting, their effectiveness as a procoagulant issignificantly impaired.

By loading the microspheres onto the chitosan fleece by methods of thepreferred embodiments, the microporous polysaccharide microspheres canbe combined with the chitosan fleece without substantial modification ofthe individual components morphologic appearances. A more evendistribution of microporous polysaccharide microspheres on the chitosanfleece can be achieved by applying the weak acid more evenly, e.g.,applying the weak acid in a smaller droplet size, such as by an atomizeror spray jet, to chitosan fiber in a rotating drum, on a conveyor belt,or the like, optionally with a vacuum source applied to the chitosanfiber to pull the droplets into the mass of fiber, and/or an air blowerto force the droplets into the mass of fiber. Tighter control ofmoisture levels during the coating process can also achieve betterdistribution of the microspheres, higher loading levels, or reducedimpact on morphology of the fleece and microspheres.

In the procedures of the particularly preferred embodiments forpreparation of chitosan fleece loaded with microporous polysaccharidemicrospheres, in order to avoid dissolution of the microspheres inaqueous solution and disruption of the microspheres' structure, only asmall amount of acidic aqueous solution or glacial acetic acid issprayed onto the surface of the chitosan fleece to maintain propermoisture levels. Consequently microporous polysaccharide microspheresare primarily “glued” onto the chitosan fleece.

While not wishing to be bound to any particular theory, is believed thatchitosan fleece, optionally loaded with microporous polysaccharidemicrospheres, can have two primary mechanisms of action when employed inwounds. One is to stop bleeding very rapidly because microporouspolysaccharide microspheres can rapidly absorb water in blood. Anotheris to repair the wound and any damaged blood vessels. The first actioncan cause the wound to stop bleeding instantly, while the secondfunction can protect the wound and prevent secondary bleeding fromdamaged or cut blood vessels. It is believed that formation of achitosan aqueous gel supported by moist chitosan fleece in the wound cancontribute to the action.

Manufacturing Processes for Loading Chitosan Fleece

As discussed above, treating chitosan by carding or similar processescan create a layer of fleece which can be disposed on a conveyor beltfor further processing. Such further processing can include loading thechitosan fleece with a polysaccharide, such as microporouspolysaccharide microspheres or other polysaccharide agents and/orhemostatic agents. In one embodiment, a method of loading chitosanfleece with microporous polysaccharide microspheres comprises coatingthe fleece with an acid, such as glacial acetic acid, optionallyfollowed by a process of applying a polysaccharide powder or otherauxiliary agent.

In an acid coating process, a weak acid is atomized then applied tochitosan fleece. This can be accomplished by any suitable methodincluding, for example, ultrasonic atomization. The atomized acidcomprises a mist or fog of acid which is passed through the chitosanfleece which rests on a conveyor. Preferably, the atomized acid mist issimultaneously blown onto the fleece by a fan and sucked through thefleece and conveyor by an extracting pump. As such, the volume of acidmist that passes through and coats the fleece can be closely controlledand the acid mist can be contained within the acid coating process area.

After the fleece has been acid coated, it can enter a powder applicationprocess area where powderized microporous polysaccharide microspheres orother powderized agents are loaded onto the fleece, if desired.Preferably, powderized microporous polysaccharide microspheres aredisposed in a vibrating sieve and are simultaneously blown onto andsucked through the fleece and conveyor by a fan and extracting pump. Assuch, the volume of polysaccharide powder applied to the fleece can betightly controlled and the polysaccharide powder can be substantiallycontained within the powder application process area.

Preferably, the acid coating process area and powder application processarea are substantially shielded from one another. After application ofpolysaccharide, the fleece preferably is delivered to a dryingapparatus.

The method as described above provides a layer of fleece loaded withmicroporous polysaccharide microspheres. It is to be understood thatfurther processing can be employed to incorporate such a layer into anonwoven fabric in which several loaded fleece layers are attached toone another to make a nonwoven fabric of polysaccharide loaded chitosanfleece.

In accordance with one embodiment, a nonwoven fabric manufacturingprocess utilizes a drum about which a backing paper is wrapped. Thebacking paper preferably is coated with acid, such as glacial aceticacid or a weak acid solution, so that a base layer of loaded chitosanfleece sticks thereto. The base fleece layer is sprayed with anothercoating of acid, and another layer of loaded fleece is placed on theacid coated fleece. Due to the acid coating on the base layer, thefleece layers fuse with one another. This acid coating and fleecewrapping process can continue for as many layers of fleece as aredesirable to create the nonwoven fabric. Once a suitable number oflayers are formed, the fleece is removed from the cylinder andtransported to a press, which exerts pressure against the fleece inorder to further bind and compact the nonwoven fabric. In oneembodiment, the fleece is dried within the press.

Chitosan with Deposited Starch

While not wishing to be bound by any particular theory, it is believedthat the hemostatic efficiency of chitosan when combined with starch ismuch better than that of the chitosan or starch alone due to theformation of a new polymer wherein chitosan intercalates with thestarch. Chitosan and starch have a similar structure and good mutualcompatibility. The interpenetration and entanglement of chitosan andstarch macromolecular chains is believed to occur when the polymers areblended with each other in a melting state or a plasticized state in thepresence of an acidic aqueous solution. However, interpenetration andentanglement of two polymer chains are generally attributed to physicalphenomenon that can alter certain physical properties of the originalpolymers while leaving their chemical structure unchanged. Consequently,interpenetrated and entangled polymers are regarded as a polymericmixture or a polymeric composite, but not a new polymer. In chitosan,there are large numbers of amino groups (—NH₂) that are able to formhydrogen bonds with oxygen atoms in a carbonyl (—C(O)—), ester (—COOR),or ether (—O—) group. It is believed that hydrogen bonding existsbetween chitosan and starch polymeric chains. However, hydrogen bondingis generally classified as a physical interaction that does not changethe chemical structure. Under certain conditions (such as heating,presence of a catalyst, and the like), an amino group can react withactive aldehyde or ketone groups to yield a Schiff linkage, which isregarded as a chemical reaction. Creation of a Schiff linkage isgenerally not observed for the combination of chitosan andpolysaccharide, since there are generally not enough active aldehyde andketone groups in the polysaccharide to facilitate such linkages.

The rheology and microscopic topology of entangled polymeric liquids wasreported by Everaers et al., Science, Vol. 303, 6 Feb. 2004, 823-826. Itwas found that the viscoelastic properties of high molecular weightpolymeric liquids are dominated by topological constraints on amolecular scale. In a manner similar to that of entangled ropes, polymerchains can slide past but not through each other. Tube models of polymerdynamics and rheology are based on the idea that entanglements confine achain to small fluctuations around a primitive path that follows thecoarse-grained chain contour. A microscopic foundation for thesephenomenological models is provided by Everaers et al., who analyze thetopological state of polymeric liquids in terms of primitive paths, toobtain parameter-free, quantitative predictions for the plateau modulus,which agree with experimental data for all major classes of syntheticpolymers.

As discussed by Viyoch et al., International Journal of CosmeticScience, 25(3), 113 (2003), chitosan with a molecular weight of 100,000and a starch such as corn, potato, or tapioca starch can be used inpreparation of a cosmetic patch containing tamarind fruit extract. Thephysicochemical characteristics, i.e., flexibility, color, transparency,integrity, gloss, water sorption, bioadhesion property, and stability,of the patch without tamarind content were investigated. A stabilitytest was performed by keeping the prepared patches at 4° C., at roomtemperature, or at 45° C. for two weeks. The results showed that theformulations composed of chitosan:corn starch ratio of 4.5:0.5(CC_(4.5:0.5)) and chitosan:tapioca starch ratios of 4.5:0.5(CT_(4.5:0.5)) and 4.0:1.0 (CT_(4.5:1.0)) provide patches with favorablephysical characteristics, high water sorption, good bioadhesion abilityand good stability. After the lyophilized tamarind extract in an amountcorresponding to 5% of tartaric acid was incorporated into theformulations of CC_(4.5:0.5), CT_(4.5:0.5), and CT_(4.5:1.0), theability of the patches to adhere to skin was improved. However, afterkeeping the test patches at room temperature or at 45° C. for 6 weeks,their colors were intensified while their flexibilities and skinadhesion properties decreased. A 12 hour in vitro permeation test wasconducted by observing the cumulative amount of tartaric acid permeatedthrough the Silastic® membrane (Dow-Corning, Midland, Mich.). TheCC_(4.5:0.5) patch tended to give the highest amount of tartaric acidreleased. The release pattern of all the blended polymeric matrices wasexhibited in two distinct phases: the rapid phase, wherein the fluxaveraged 3.61 μg min⁻¹ mm⁻²; and the slow phase, wherein the fluxaveraged 1.89 μg min⁻¹ mm⁻². The methods described by Viyoch et al. forpreparing blends of chitosan and starch can be adapted for preparationof chitosan substrates or hemostatic agents suitable for use inpreparation of materials of preferred embodiments.

Chitosan with Deposited Dextran

Superior coating can be achieved by providing the chitosan fibers with adextran coat having a microporous surface, rather than by depositingmicroporous polysaccharide microspheres. Such a dextran coating can beprepared in any number of ways. For example, dextran can be solubilizedin water or a mild acid solution to confer electronegativity to themolecules by mild oxidation, whereby carboxyl groups are created. Thedextran can also be reacted under controlled conditions to entangle thepolymers. The chitosan dextran mixture can be lyophilized to create asurface that has microporosity on the chitosan fibers. The degree ofdextran coating can be controlled, the thickness of the coating can becontrolled, and the porosity size can be controlled by the variouscontributions of either a straight or branched chain amylose oramylopectin. A chemical bonding can take place with the reaction of anegatively charged dextran and the cationic chitosan. All of theseprocesses can be monitored by both light and electron microscopy as wellas by histochemistry of sections of the material to determine whethersuccessful bonding has been achieved.

A variation of this technology involves preparing alternate layers ofdextran-coated microporous chitosan and non-coated chitosan to prepare agradient of a non-woven fleece and fabric. Methods known in the art forapplying dextran coating to various substrates can be modified to yieldmethods of applying dextran coatings to chitosan fibers. Microporouspolysaccharide microspheres can also exhibit superior adhesion todextran-coated chitosan fibers. Accordingly, in certain embodiments itcan be desirable to pretreat chitosan fibers with dextran prior todeposition of the microporous polysaccharide microspheres.

Chitosan-Microporous Polysaccharide Microsphere Production Process

The term “chitosan” corresponds to a family of polymers that vary indegree of N-deacetylation (DA). Chitosan generally varies from about 50to 70% DA or higher with variable viscosity, solubility, and hemostaticproperties. Since the behavior of chitosan polymers, namely theirreactivity, solubility, and ability to bind MPH, depends on the DA ofchitin and chitosan, an assay to determine DA is desirable. FTIRspectroscopy and C13 mass NMR spectroscopy are linked for chitosanassays. The technique used to determine the degree of acetylation ofchitin and chitosan is preferably FTIR spectroscopy. FTIR spectroscopyhas the advantage of being nondestructive, fast, extremely sensitive,user friendly, low priced, and applicable for both soluble andnonsoluble samples.

Prior to assay, all proteins and endotoxins are removed from the chitinas produced for clinical applications. Chitosan fibers are examined todetermine their cross section, their tensile strength, breakingstrength, loading strength, and their appearance. This industrialengineering process is utilized in the manufacture of chitosan fleeceand chitosan sponge, as well as in the manufacture of chitosan fabric.The amount of saturation of microporous polysaccharide microspheres istested in model systems to determine appropriate physicalcharacteristics for three major types of bleeding.

Characterizing the Structure and Properties of the Chitosan Fiber

Established and on-line methods for measuring the crystal structure,size, chitin DA, average molecular weight, content of heavy metals, andtoxicity of the chitosan fiber are employed. Characterization testingincludes testing for fiber strength, pulling rate, mean fiber swellingas ratio of fiber diameter after absorption to that before absorption ofdistilled water, and pH. Chitosan having a DA of 50 to 80 wt. % or moreis compared. Materials that are assayed include microporouspolysaccharide microspheres, chitosan of varying DA, andchitosan-microporous polysaccharide microsphere materials. Measurementsof water and blood absorption, rates of water and blood release, localretention (using gel strength), and screening tests for hemostasis arealso conducted. Since erythrocyte polymerization (agglutination) isconsidered a major factor for chitosan-induced blood coagulation, asimple hemagglutination test can be used for rapid screening of theproduct.

Simple hemagglutination assays are known in the art. Chitosan,chitosan-microporous polysaccharide microsphere, and microporouspolysaccharide microsphere compositions are prepared in stock solutionscontaining 2000 μg/ml of the material. A 10 fold dilution is used toachieve a final concentration of 1000, 100, 10, or 0.1 μg/ml in a volumeof 0.2 ml in 0.9% NaCl (normal saline). Human red cells (obtained from ablood bank) are rinsed twice with Alsever's Solution and twice with 0.9%sodium chloride. Sodium chloride is used to circumvent incompatibilitybetween deacetylated chitin and other ions. Washed red cell aresuspended in a saline solution (0.9% NaCl) and adjusted to 70%transmission with a colorimeter (Klett-Summerson, No. 64 filter). Anequal volume of red cell suspension (0.2 ml) is added to the variousdilutions of chitosan-microporous polysaccharide microsphere, chitosan,and microporous polysaccharide microsphere compositions. Tubes areincubated for 2 hours at room temperature before reading. Deacetylatedchitin (chitosan) normally produces hemagglutination of human red bloodcells at a concentration of 1 μg/ml.

Protein binding capacity can be determined using biomedical sensorsutilizing reflectometry interference spectroscopy (RIFS), that enablesthe kinetics of the absorption of proteins onto the surface of chitosan,chitosan-microporous polysaccharide microsphere, and microporouspolysaccharide microspheres alone to be determined. Once an optimalchitosan-microporous polysaccharide microsphere is reached forhemostasis, batches can be quickly evaluated for protein bindingcapacity and this parameter related to hemostatic effectiveness in therat model cited above.

Optimization of microporous polysaccharide microsphere loading tochitosan can be achieved using systems other than the acetic acidtreatment for solution and loading microporous polysaccharidemicrospheres into the chitosan. Lactic acid can reduce more toxicitythan acetic acid. The binding of microporous polysaccharide microspheres(starch), a non-polar polysaccharide to chitosan—a strongly cationicpolysaccharide can conceivably be enhanced by selective starchoxidations and generation of an anionic state.

Studies of the degradation kinetics of chitosan fibers, chitosan fleece,and fabric, but with and without microporous polysaccharide microspheresare conducted. Studies of the hemostatic mechanism ofchitosan-microporous polysaccharide microsphere fleece and fabric areconducted using multi-photon imaging and spectroscopy to evaluate themolecular interaction of chitosan, chitosan-microporous polysaccharidemicrosphere, and microporous polysaccharide microspheres with human andporcine whole blood and platelets. These results are compared to thedeterminations offered by application of NAG. In vitro clot formation,RBC aggregation, and platelet activation are studied.

Design and manufacture of a production line for large scale productionof microporous polysaccharide microsphere mixed chitosan fleece andnon-woven fabric is conducted. Machines to perform the followingfunctions are developed to loosen the chitosan fiber; to card theloosened fiber into a thin fleece net; to moisten the chitosan fiberfleece by dilute acetic acid (or lactic acid) solution; to homogeneouslyload microporous polysaccharide microspheres onto the thin piece ofmoist chitosan fiber; to roll up the thin piece of microporouspolysaccharide microsphere-loaded chitosan fiber on a reel; and to drythe fiber in a vacuum. A fully automatic or semi-automatic productionline is designed and assembled to produce a standardized bulk quantityof chitosan-microporous polysaccharide microsphere fleece and nonwovenfabric. Tests of the density of varied fleece preparations are conductedto optimize interstice size and optimal fleece density for hemostasis.Similar tests are performed on collagen fleece.

Optimizing Chitosan-Microporous Polysaccharide Microsphere Formulationsto Meet the Needs of Specific Hemorrhagic Diathesis

Formulations are optimized using models that the military has definedfor testing and comparative evaluation of chitosan-microporouspolysaccharide microsphere materials. These models include the fatalaortic punch lesion and large venous and diffuse capillary bleeding in aliver injury models (swine). The model for remote closure of arterialcatheterization lesions is taken from the literature and can be readilyadapted to close lesions with chitosan-microporous polysaccharidemicrospheres. The oral bleeding model in the rabbit permits testing in avascular organ system in an animal whose coagulation status can bereadily modified (platelets, heparinization). This model has been testedwith liquid chitosan as a hemostatic agent.

Animal Hemostatic Testing of the Chitosan-Microporous PolysaccharideMicrosphere Fleece and Fabric

Hemostatic tests have been performed on injured large vessels (e.g.,catheterized canine femoral artery) under heparinization, and also tothe lethal swine transection model (femoral artery, femoral vein), andthe punctured rat femoral artery and vein model.

Catheterized Canine Femoral Artery

The model for control of brisk bleeding after arterial puncture andcatheterization is the puncture of the canine femoral artery inheparinized animals. Three animals had an 11.5 French catheter placedfor 4-6 hours in the femoral artery, then were heparinized withactivated clotting times (ACT) 2-3 times normal, and were maintained atnormotensive levels by IV fluid replacement. The indwelling arterialcatheter was removed and a chitosan-microporous polysaccharidemicrosphere patch (2×2 cm) was immediately applied to the bleedingvessel with minimal pressure for 10 minutes. Videotapes documented thesestudies.

Tests were conducted on Dog Three having a weight of 25.7 kg; sex F; andcoagulation time ACT 277 seconds. The catheter in the dog femoral arterywas a 11.5F catheter. 1-2 cm³ of chitosan-microporous polysaccharidemicrosphere fleece was placed on the femoral artery puncture holeimmediately after the 11.5 F catheter was removed. Manual pressure wasapplied on the fleece for 10 minutes and bleeding was completely stoppedwith absolute hemostasis. A chitosan-microporous polysaccharidemicrosphere patch was applied to a femoral vein puncture hole, another11.5 F catheter was removed, and held with manual pressure for 7minutes. Complete hemostasis was achieved. Venous pressure was increasedby proximal ligation and the chitosan-microporous polysaccharidemicrosphere patch adhered without bleeding.

Tests were conducted on Dog Four having a weight of 25.4 kg; sex F; andcoagulation time ACT 280 seconds. A 1-2 cm³ chitosan-microporouspolysaccharide microsphere patch was placed on the femoral arterypuncture hole immediately after the 11.5 F catheter was removed, andmanual pressure was applied for 10 minutes. Complete hemostasis withmarked adherence of the fleece was noted.

Tests were conducted on Dog Five having a weight of 23.1 kg; sex M; andcoagulation time ACT 340 seconds. PVA treated chitosan-microporouspolysaccharide microsphere fleece (1 cm³) was applied to the femoralartery puncture hole after the 11.5 F catheter was removed and manualpressure was applied for 10 minutes. Bleeding stopped, but 30 secondslater moderate bleeding from the puncture wound was noted. A secondattempt using the same PVA treated chitosan-MPH fleece (with 10 minutesmanual compression) failed. Chitosan-MPH non-woven fabric without PVAwas then used to replace the relatively non-adherent PVA treatedchitosan-MPH fleece. Complete hemostasis was achieved after 15 minutesof manual compression. The wound was observed for 20 minutes and nobleeding was noted. The non-PVA treated chitosan-MPH fabric adheredtightly to the artery and surrounding tissue. Artery with fabric wasremoved for pathology.

The dog experiments demonstrated that chitosan-microporouspolysaccharide microsphere fleece (non-PVA treated) was remarkablyeffective as a hemostatic agent in the heparinized canine arterialcatheterization model. Large bore catheter (11.5 F), left in place for4-6 hours results in a significantly molded, vascular breech and in theface of significantly prolonged coagulation time represents asubstantial hemostatic challenge. Chitosan-MPH fleece also conforms tothe arterial contour, does not interfere with distal flow, and isremarkably adherent. Chitosan-microporous polysaccharide microspherefleece was equally effective in achieving hemostasis in the catheterizedfemoral vein and was also remarkably adherent without interfering withflow. Chitosan-microporous polysaccharide microsphere fleece (PVAtreated) achieved moderate to minimal hemostasis in one trial, and wasrelatively non-adherent. Complete hemostasis was secured with a non-PVAtreated chitosan-MPH fabric patch.

Rat Punctured Femoral Artery and Vein

Femoral arteries and veins of three rats (OD 1.5 to 2 mm) were exposedbilaterally after barbiturate anesthesia was achieved. Puncture woundswere made in each artery with a 30 gauge needle, and a pledget (3 mm³)of chitosan-microporous polysaccharide microsphere fabric was placed onthe puncture site for 10 seconds and monitored for bleeding. PVA treatedmaterial was not used. Control of bleeding from the injured thin walledrat femoral vessel for 100 min. is a hemostatic challenge. Afterexposing both femoral arteries, a 30 gauge needle was used to puncturethe arteries to create an arterial laceration and brisk bleeding.

Tests were conducted on Rat No. 1, a male weighing 520 g. The rightfemoral artery puncture wound was treated with a pledget ofchitosan-microporous polysaccharide microsphere fabric. Gentlecompression was applied to the pledget for 30 seconds, and after releasethere was very slight bleeding under the fabric. Gentle manual pressurewas applied again for 10 seconds and the bleeding completely stopped.After 20 minutes observation of complete hemostasis, both proximal anddistal ends of the femoral artery were ligated and a burst strength testwas conducted. The fabric repaired wound remained intact at 120 mm Hg.

Tests were conducted on Rat No. 2, a male weighing 525 g. The leftfemoral artery puncture wound was treated with a 3 mm² pledget ofchitosan-microporous polysaccharide microsphere fabric. Manualcompression was applied on the fabric for 10 seconds. After release ofmanual pressure there was slight bleeding under the fabric patch. 2seconds of additional manual pressure was applied but minimal bleedingcontinued at a diminishing rate. No additional pressure was applied andbleeding stopped completely after 56 seconds. After 20 minutes ofcomplete hemostasis both proximal and distal end of the femoral arterywere ligated and the burst strength test was conducted.Chitosan-microporous polysaccharide microsphere fabric repaired woundwithstood arterial pressure until 300 mm Hg. The right femoral arterypuncture wound was treated by placement of a fat pad over the injury.Manual compression was applied on the fatty tissue for 10 seconds. Afterrelease of the manual pressure there was profuse bleeding under thefatty tissue. No additional pressure was applied. The bleeding stoppedafter one minute and 27 seconds and 20 minutes later both proximal anddistal end of the femoral artery were ligated and a burst strength testwas conducted. The fatty tissue repaired wound failed at approximately60 mm Hg.

Tests were conducted on Rat No 3, a male weighing 555 g. A right femoralartery puncture wound was treated with a chitosan-microporouspolysaccharide microsphere 3 mm² pledget mixed chitosan non-woven fabricused to cover the wound. Manual compression was applied for 20 seconds,and after release complete hemostasis was secured. After 20 minutes ofobservation, both proximal and distal ends of the femoral artery wereligated and a burst strength test conducted. The chitosan-microporouspolysaccharide microsphere patch withstood arterial pressure until 200mm Hg. The right femoral artery puncture wound was covered with fattytissue. Manual compression was applied on the fatty tissue for 20seconds and after release of the manual pressure there was profusebleeding. Bleeding stopped after one minute and 21 seconds withcontinued manual pressure. After that, both proximal and distal ends ofthe femoral artery were ligated and a burst strength test was conducted.The fatty tissue patch failed at less than 120 mm Hg (approx. 60).

The rat tests demonstrated that chitosan-microporous polysaccharidemicrosphere pledgets were remarkably effective in achieving completehemostasis in the face of brisk bleeding from a puncture wound in afragile vessel. The time required for the chitosan-microporouspolysaccharide microsphere fabric to stop bleeding varied from 20seconds to 56 seconds. This could be caused by an uneven distribution ofmicroporous polysaccharide microspheres on the chitosan non-woven fabricpatch. The chitosan-microporous polysaccharide microsphere patch adheresvery tightly to the vessel and can withstand high arterial pressuresbefore failing. The rat femoral artery puncture model is an excellentscreening system to study mechanisms for hemostasis and tissue adherenceas well as screening of various chitosan-microporous polysaccharidemicrosphere formulations.

Swine Femoral Artery and Femoral Vein

Tests were conducted wherein a lethal large artery injury transects thefemoral artery and femoral vein. The chitosan-MPH puff providedremarkable hemostasis in comparison to other methods that were utilized.

Fatal Aortic Injury Model in the Pig

This model was developed for hemostatic agent testing conducted at theU.S. Army Institute of Surgical Research, San Antonio, Tex., for thepurpose of determining the optimal hemostatic dressing for high pressurearterial bleeding. The injury is a calibrated puncture hole in thedistal aorta of normotensive pigs. Nine different hemostatic dressingswere evaluated for this otherwise 100% fatal injury. The only animalsthat lived 60 minutes received the American Red Cross Fibrin Dressing(Fibrin and Thrombin) or had suture repair of the lesion. All otherhemostatic agents to include NAG failed to control the aortal hemorrhageand no animals survived 60 minutes. Chitosan and microporouspolysaccharide microsphere were not included in these experiments.

Five groups of five pigs (40 kg, immature Yorkshire cross swine male)restudied. One group is treated with American Red Cross Fibrin Dressing,the other four groups with chitosan fabric with or without microporouspolysaccharide microspheres and chitosan fleece with or withoutmicroporous polysaccharide microspheres. Microporous polysaccharidemicrospheres alone generally do not control brisk arterial bleeding andare not included. Previous experiments demonstrated the fatality of thelesion untreated and that the animals can be rescued by suture repair.The objective of this study is to compare the American Red CrossDressing to chitosan-based dressings. Survival, blood loss, and amountof IV resuscitatable fluid to maintain normotension are determined.

Animals are premedicated (Telazol 4-6 mg/kg IM, Robinul 0.01 mg/kg 1M),endotracheal anesthesia is maintained with 1-3% isofluorane and oxygen,and core temperature held between 37°-39° C. Indwelling arterial linesare placed for both proximal (carotid) and distal (femoral) MAP (MeanArterial BP determinations) and a femoral IV line is inserted forresuscitative fluid administration. Pigs are spelenectomized, the spleenweighed, and replacement fluid (3× splenic weight of warm lactatedRingers) solution administered to correct for blood removal (spleen).

Hemodynamic stabilization is secured after splenectomy within 10 minutesand arterial blood samples (12 ml) are obtained prior to the aorticpunch. The aortic injury is made immediately after aortic occlusion andarterial blood is drawn 30 and 60 minutes after the injury. Prothrombintime, activated partial thromboplastin time, fibrinogen concentration,thromboelastogram, complete blood count, lactate, and arterial bloodgases are determined.

After the splenectomy and 10 minute stabilization period, drains tocontinuous suction are positioned bilaterally in the lateral abdominalrecesses. Rate of bleeding is determined by weighing the blood loss overtime and is expressed as grams accumulated per 10 seconds. After crossclamping the aorta above and below the site of the injury, (3 cm abovethe bifurcation of the distal aorta, aortotomy made with a 4.4 mm aortichole punch) cross clamps are removed. Bleeding is initially tamponadedby placing a finger on the hole without vessel compression. At 0 timethe finger relieves the tamponade and brisk arterial bleeding is allowedfor 6 seconds. Blood is collected and rate of blood loss monitored bydeflecting blood into the peritoneal cavity for drainage.

A polyethylene elastic sheet is placed between the dressing and glovedhand and after 6 seconds of brisk bleeding the test hemostatic dressingis applied for four minutes. Manual compression consists of completeaortal occlusion as manifested by a non-pulsatile femoral BP (MAP at 15mm Hg). After four minutes, manual compression is relieved leaving thedressing and plastic sheet over the injury site. The injury site isobserved for bleeding for two minutes. A key endpoint is a completeabsence of bleeding after 2 minutes of observation. If bleedingpersists, another four minutes of compression is administered. In theevent of active bleeding or no hemostasis, resuscitation is discontinuedand the animal is allowed to die. In order to test the adherence of thetest dressing with no evidence of bleeding, resuscitation is institutedwith 37° C. lactated Ringer solution at a rate of 300 ml/min IV. Apre-aortotomy baseline MAP plus or minus 5 mm Hg is maintained for anadditional 60 minutes. Death (a key endpoint) is a MAP <10 mm Hg and endtidal PCO2 less than 15 mm Hg. At the end of the experimental period(euthanasia at 1 hour in surviving animals) aortas are removed, opened,and evaluated. After the lesion is observed and photographed the size ofthe hole is measured to ensure uniformity of injury size, and thespecimen fixed for histological examination to evaluate the hemostaticprocess (fibrin, platelets, extension into lumen).

Though the ARC hemostatic dressing has provided survival in this modelit still has disadvantages. The “ideal” hemostatic dressing, in additionto the parameters cited earlier, controls large vessel arterial venousand soft tissue bleeding, adheres to the vessel injury but not to theglove or hands, is flexible, durable, inexpensive, stable in an extremeenvironment, has a long shelf life, does not require mixing, poses norisk of disease transmission, does not require new training, and ismanufactured from readily available materials. None of the dressingsthat have been tested or evaluated in the current setting meet all ofthese characteristics. The shortcoming of the fibrin-thrombin AmericanRed Cross field dressing (ARC) is that it is fragile in its currentform. The field dressing is stiff and thick when dry and some of thelyophilized material flakes off when the field dressing is grasped. Thefibrin-thrombin dressing sticks to latex gloves and skin when wet. Thehandling characteristics of the chitosan fleece with MPH are superior tothese prior art materials.

The Canine Femoral Artery Catheterization Model

Extensive background literature for the evaluation of this novelvascular sealing device exists. Femoral arteries are studied bypercutaneous placement of standard vascular sheaths (7 French) withcatheters inserted by the Seldinger technique. A total of twenty animalsare utilized, ten anticoagulated with IV heparin 150 units/kg toactivated clotting times (A.C.T.) 3× normal. The ACT is measured justprior to insertion of the sealing device. Unheparinized animals have thecontralateral femoral artery used as a control with only manualcompression used to achieve hemostasis. Arterial sheaths and cathetersare left in place for one hour to simulate an intervention duration. Thevascular sealing device with the chitosan-MPH in one femoral artery andmanual compression are utilized on the other femoral artery. Manualpressure applied to the puncture site is released and the puncture siteis inspected every five minutes for the following key endpoints:external bleeding or hematoma formation, measurement of thighcircumference, integrity of the distal pedal pulses, and manualcompression time required to achieve hemostasis. Animals are observedfor an additional ninety minutes, then euthanized with an overdose of IVsodium pentobarbital and saturated potassium chloride. Prior toeuthanization, animals are subjected to femoral angiography in eachgroup.

A subgroup of animals survive with a follow-up examination at 2 weeks.This includes physical inspection of the arterial access, assessment ofthe distal pulses, femoral angiography, and histopathologic examinationof the excised femoral artery puncture site and surrounding tissue.Statistical analysis is expressed as mean standard deviation. Thestudent's t-test unpaired is used for comparing the mean times tohemostasis within the different treatment groups. Preliminary animalstudies are performed before proceeding to human clinical trials.Chitosan fleece with microporous polysaccharide microspheres andchitosan fabric with microporous polysaccharide microspheres bothexhibit superior performance in controlling blood loss as well as theother parameters tested.

Model for Severe Large Venous Hemorrhage and Hepatic Injury (Swine)

This model has been extensively tested by the U.S. Army Combat CasualtyCare Research Program. There is a large baseline of data regardingextent of injury and response to a variety of hemostatic agents. Thisdata includes documentation of the extent of injury to large diameterveins, ability to apply hemostatic dressings in the face of massivebleeding, extent of blood loss, facility of instrumentation, lethality,and reproducibility of the experimental liver injury. Both the AmericanRed Cross Hemostatic Dressing (ARC) and the experimental chitosanacetate sponge proved to be effective hemostatic agents in this model.The military investigators concluded that both the chitosan and AmericanRed Cross Dressing (ARC) warranted further studies and development. Thehemostatic effectiveness of chitosan (fleece, fabric, with or withoutmicroporous polysaccharide microspheres) and the ARC dressing in the pigsevere large venous hemorrhage model was tested. The recommendedconventional therapy for treating Grade V hepatic injuries (extensiveparenchymal damage combined with major vascular lacerations) istamponade with gauze sponges and later reoperation. The issue ofbiodegradability and wound healing has never been resolved with thesehemostatic agents. Consequently surviving animals are sacrificed onemonth post-injury to examine the healing wound and hemostatic agentdegradation. Hemostatic control is monitored over the one-month periodby weekly hepatic CT scans. Evidence of rehemorrhage requiresintervention laparotomy and animal sacrifice. The post-injury andhemostatic repair course of the animals is monitored.

Crossbred commercial swine (males, 40-45 kg) are divided into six groupsof five animals each. Test groups consist of gauze packing, ARCdressing, chitosan fleece with or without microporous polysaccharidemicrospheres and chitosan fabric with or without microporouspolysaccharide microspheres. Surgical preparation and anesthesia is asfor the aortic punch injury model. Carotid artery and jugular vein linesare placed, and splenectomy and urinary bladder catheter placementcompleted. Both hemodynamic (stable MAP for 15 minutes) and metabolic(rectal temperature 38-40° C., arterial blood pH 7.39-7.41)stabilization are achieved. Arterial blood samples are obtained. Eachtest animal must have a normal hematocrit, hemoglobin concentration,platelet count, prothrombin time, activated partial thromboplastin time,and plasma fibrinogen concentration to be included in the study. Drainsare placed bilaterally (as in the aortotomy) for rate and quantitativeblood loss calculation. The liver injury is induced exactly as describedin previous publications. Essentially, a specially designed clamp, “X”shaped, consisting of 4.5 cm sharpened tines and a base plate is used tomake two penetrating liver lacerations. The standardized liver injuriesare through-and-through stellate wounds, involving the left medial lobarvein, right medial lobar vein, portal hepatic vein, and hepaticparenchyma. Thirty seconds after injury, warm (39° C.) lactated Ringer'ssolution is started at a rate of 260 ml/min to restore the baseline MAP(Mean Arterial Pressure). The experimental hemostatic dressings areapplied at the same time as IV fluids are initiated with manualcompression via standardized applying pressure in a dorso-ventraldirection. After one minute, the wound is inspected for bleeding. Ifhemostasis is not complete, pressure is reapplied in the lateromedialdirection. The sequence is repeated four times, with 60 seconds ofcompression. The key endpoints of hemostasis are defined as the absenceof any detectable bleeding from the injury. After application of thehemostatic treatment, the animals' abdomen is temporarily closed and theanimal observed for 60 minutes. The endpoint for death is a pulse of 0.Quantitative blood collection prior to treatment application is termed“pretreatment blood loss,” at the end of the study period this isreferred to as the “post-treatment blood loss.” Blood in the hemostaticagents is not included but total IV fluid replacement and estimatedpre-injury blood volume is determined.

Adherence strength of the hemostatic dressing is estimated using thesubjective scoring system reported by the military team who devised thisprotocol. Scores range from 1 to 5; 1=no adherence, 2=slight,3=adherence to cause stretching of tissue in contact with hemostaticagent but not lifting liver from table, 4=adherence sufficient topartially lift liver from table, and 5=sufficient adherence to liftliver from table. The mean score for the 3 dressings from each animal istreated as a single value for adherence strength.

Key endpoints are survival, death, pretreatment blood loss,post-treatment blood loss, survival time, hemostasis at 1, 2, 3, or 4minutes, % resuscitation fluid volume, (no values for adherence datagiven). Key parameters of injury are number of vessels laceratedcorrelated with pre-treatment blood loss in ml and ml/kg body weight.

Chitosan fleece with microporous polysaccharide microspheres andchitosan fabric with microporous polysaccharide microspheres bothexhibit superior performance in controlling blood loss as well as theother parameters tested.

Oral Bleeding Model: Lingual Hemostasis in the Rabbit

This model provides convenient hemostatic testing in a system withenhanced capillary blood flow (the tongue) and high fibrinolyticactivity (oral mucosa). This model can easily have platelet functionsuppressed as well as be heparinized. The model has been used toevaluate the hemostatic effect of liquid chitosan in dilute acetic acidwith the key endpoints of a reduced bleeding time after a standardincision. Descriptions of the model have been published and providebaseline data for the results to be compared.

The hemostatic effectiveness of NAG, considered highly hemostatic forcapillary hemorrhage, is compared with chitosan fleece with and withoutmicroporous polysaccharide microspheres and chitosan fabric with andwithout microporous polysaccharide microspheres. The key endpoints arelingual bleeding time, measured in minutes from the time the hemostaticagent is applied until hemostasis is complete. Rabbits are euthanizedone to fourteen days after the surgery and the lesions evaluatedhistologically. Rabbits with normal blood coagulation status, suppressedplatelet activity, and heparin anticoagulation are studied. NZW rabbits,5-6 lbs, are studied for lingual hemostasis after using the modeldeveloped by Klokkevold, et al., consisting of a special metal stentsutured to the tongue in order to stabilize soft tissues and insure aconsistent injury. Tongue incisions on the lateral border are made witha guarded 15 blade knife. Bleeding time measurements from the incisionare made using the filter paper procedure of Coles. Blots are takenevery 15 seconds until no blood staining occurs. Systemic bleeding andcoagulation times are also determined. A total of 30 rabbits arestudied, six groups of five each. The six groups consist of control (notreatment), NAG, chitosan fleece with or without microporouspolysaccharide microspheres, and chitosan fabric with or withoutmicroporous polysaccharide microspheres. After animals are anesthetized(IM Ketamine HCl 35 mg/kg and Xylazine 5 mg/kg) an ocular speculum isinserted into the mouth to hold it open and the stainless steel stentsutured to the tongue to stabilize tissues. Tongue incisions are madewith a depth of 2 mm, length 15 mm on the lateral border of the tonguewith a guarded 15 blade. Incisions are immediately treated with thehemostatic agents and bleeding times measured. The method of tonguemarking prior to incision is utilized to facilitate histologicsectioning post-marker. The identical study as above in 30 rabbits, fivegroups of five each, is conducted in animals treated with a plateletfunction antagonist—epoprostanol (prostacyclin or PGI₂). The protocol ofKlokkevold is followed explicitly. Again, 30 rabbits are studied afterthe activated coagulation time has been prolonged 3× as well asincreasing the mean systolic bleeding time by 40%. The histological examis to include SEM. Chitosan fleece with MPH and chitosan fabric with MPHboth exhibit superior performance in controlling oral bleeding.

Swine Tests—Lethal Groin Injury Model

1.5 g of chitosan fleece loaded with microporous polysaccharidemicrospheres was tested in one swine (94 kg) in the lethal groin injurymodel developed by Dr. Hasan Alam, a trauma surgeon and researchspecialist in the area of battlefield casualties who is on the staff ofthe Washington (DC) Hospital Center and serves on the faculty of theUniformed Services University of the Health Sciences (USUHS). Aftersevering the femoral vessels, bleeding was allowed to continue until theanimal had lost blood equivalent to approximately 20 to 25 ml/kg of bodyweight. By allowing a fixed amount of blood loss based on body weightrather than bleeding for a specific time, a more consistent pathology iscreated. After the fixed amount of blood loss, a 1.5 g piece of chitosanfleece was placed on the wound. The chitosan fleece was successful incontrolling the bleeding from the severed vessels. Followingresuscitation with 500 ml of Hespan, the animal was observed until themean arterial pressure had stabilized at approximately 45 mm Hg. Nobleeding was observed in this observation period. At that time,additional resuscitation plus calcium and dopamine were administered toincrease the mean arterial pressure to 95 mm Hg with no indication ofbleeding. The animal was sacrificed and the fleece was examined.

Altered chitosan fleece was tested on another swine (87 kg). Thechitosan fleece was altered by adding additional microporouspolysaccharide microspheres to the fleece to compensate for any loss ofmicroporous polysaccharide microspheres during the alteration. Theapplication was successful and after approximately one hour the meanarterial pressure was increased using Hespan and dopamine to stress theapplication. No bleeding was observed until the mean arterial pressurewas raised to approximately 95 mm Hg, at which time bleeding resumed.The fleece was removed. The material was observed to be stiff and hard.An additional fleece was placed in the wound. After that, the bleedingwas controlled. The additional fleece was unaltered chitosan fleececontaining microporous polysaccharide microspheres.

Both of these applications demonstrated that to achieve satisfactorycontrol of strong bleeding, it is necessary to place the fleece on theinjured vessel. Little bleeding occurs in the bottom of the femoralwound. If the material is placed in the bottom of the wound, bleedingmay not be controlled. Accordingly, the material is preferably carefullyplaced in the wound so as to contact the femoral artery. Overall, theapplications were very successful and suggested that when properlyapplied the formulations have good efficiency.

The above description discloses several methods and materials of thepresent invention. This invention is susceptible to modifications in themethods and materials, as well as alterations in the fabrication methodsand equipment. Such modifications will become apparent to those skilledin the art from a consideration of this disclosure or practice of theinvention disclosed herein. Consequently, it is not intended that thisinvention be limited to the specific embodiments disclosed herein, butthat it cover all modifications and alternatives coming within the truescope and spirit of the invention as embodied in the attached claims.All patents, applications, and other references cited herein are herebyincorporated by reference in their entirety.

1-20. (canceled)
 21. A hemostatic material, the material a plurality oflayers of chitosan fiber secured to each other to form a net structureby treatment with a weak acid solution of pH 3.0 to 4.5, the hemostaticmaterial further comprising microporous polysaccharide microspheresadhered to the chitosan fiber.
 22. The hemostatic material of claim 21,wherein the weak acid is acetic acid.
 23. The hemostatic material ofclaim 21, wherein the material is a nonwoven fabric.
 24. The hemostaticmaterial of claim 22, wherein the nonwoven fabric has a rough side and asmooth side.
 25. The hemostatic material of claim 22, wherein thewherein both sides of the nonwoven fabric are rough.
 26. The hemostaticmaterial of claim 22, wherein the fabric comprises from 2 to 25 layers.27. The hemostatic material of claim 21, wherein the chitosan fibers areof uniform thickness.
 28. The hemostatic material of claim 21, whereinthe chitosan fibers are of a mixture of thicknesses.
 29. A process forpreparing a hemostatic material, comprising: a) providing a first layerof chitosan fibers; b) applying a weak acid solution of pH 3.0 to 4.5 tothe first layer of chitosan fibers; and thereafter c) depositingmicroporous polysaccharide microspheres on the first layer, whereby themicroporous polysaccharide microspheres are adhered to the chitosanfibers, whereby a hemostatic material according to claim 21 is obtained.30. The process of claim 29, wherein steps a) through c) are repeated atleast once.
 31. The process of claim 29, further comprising: heating thehemostatic material, whereby a liquid is vaporized from the hemostaticmaterial.
 32. The process of claim 29, further comprising: d)compressing the hemostatic material between a first surface and a secondsurface; and e) heating the hemostatic material, whereby a dryhemostatic material is obtained.
 33. The process of claim 29, furthercomprising: placing a second layer of chitosan fibers atop the firstlayer of chitosan fibers, wherein the step is conducted after step b).34. The process of claim 29, wherein the weak acid solution is an aceticacid solution.